GRE BioChem
Transcript of GRE BioChem
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TO THE GRE BIOCHEMISTRY SU JECT TEST
n ~ g Marquart
CHEMISTRY
electron donors
electron acceptors
law of thermo energy converted, never created or destroyed
law of thermo - universal entropy always increases
projection - 2D representation of 3D structure
- same chemical bonds
mirror image steroisomers
- steroisomers that are not mirror images
- clockwise up
- counterclockwise up
- cyclic rotation about 1 axis),
dihedral rotation along 2 axis),
icosahedral- 20 faced (virus capsid), helical
energy for synth on growing polymer
proteins & fatty acids
energy for synth on added monomer
DNA, RNA, polysaccharides
lattice of one molecule trapping another
layer shell of water around hydrophobic molecule
- structural motif that binds NAD/NADP cofactors
> [NADHj
nicotinamide adenine dinucleotide
oxidations, catabolic
< [NADPH]
nicotinamide adenine dinucleotide phosphate
reductions, anabolic
flavin adenine dinucleotide
- rapid oxidation & reduction
heme - porphyrin ring + (held by 4 nitrogen)
proteins iron-sulfer center)
Q) 1 2 electrons
- from vitamin riboflavin
expresses relative contrib. of enzymes to
metabolites flow through pathway. sum of lUX control
ical catalysts
different forms of enzyme that catalyze same reaction
does not contain easily ionizable functional groups
catalytic antibody
Scatchard analysis - quantifies affinity blw receptor & ligand
TP resonance & charge on Pi contribute to high potential
phosphorylation - usually at AAs w i OH side chains (5er, Thr, Tyr)
Inhibition
Agonists - structural analogs that bind receptor & mimic effects
Antagonists anologs that bind wlo triggering normal effect
competitive inhibition - binds active site, blocking catalysis
o Vmax same, Km increases plots cross)
un competitive inhibition - bind to E5 complex at separate si
o Vmax & Km lowered plots parallel)
mixed inhibition - binds alternative site wi or wlo substrate
o effects both Vmax & Km
noncompetitive inhibition very unusual
. 0 Vmax lowered, Km same (plo ts cross)
Alloseteric Enzymes
heterotropic effects non-substrates shift sigmOidal plot to right
homotropic effects - substrates shift sigmoidal plot to left
i c h a e l i s ~ e n t e n equation
hyperbolic dependence of Va on [51
usefulfor detecting
allostericregulation
neither Vmax nor Km immediately clear
double-reciprocal plot 1/V vs 1/[5])
more accurate determination ofV max & Km
o y-intercept - l Vmax
ox- intercept l /Km
Vmax - reaction rate when all active sites saturated wi substrate
m Michaelis constant) - [5] at]l, Vmax
Keat general rate constant / turnover number) of substrate
converted to product when saturated per unit time (Vmax [enzym
eal m - specificity constant
Q mass-action ratio) - [P]/[Rj
1-K,n
ENZYMES
zymogen proenzyme - inactive enzyme precursor
Condensation Reactions
synthases - catalyze condensation wlo NTP
synthetases - catalyze condensation wi NTP
Jigases catalyze condensation of two atoms wi NTP
hydroxylase - hydroxylates substrate
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-1-2 oxygens of O2 incorporated into substrate
- both atoms of O2 incorporated into substrate
involved in nearly all aromatic ring cleavages
/ oxioreductases - catalyze redox reactions
- hydrolytic cleavage
cleave tryglycerides into glycerol & fatty acids
- interconvert stereo, structural or positional isomers
mutase - transfers functional group win single molecule
serine/threonine kinases - primarily pp-ate serine
tyrosine kinases - Src largest family of cytoplasmic TKs
function oxidase - incorporates O2 into steroids, etc
non-protein bound to enzyme
2+ - complexes w/ ATP to form substrate for kinases
2+ - often found in enzyme active site
- permanently bound cofactor
nucleotide NAD, etc
cofactor in coenzyme A, NAD+, FAD, NADP+
- organic non-protein carries groups bin enzymes
- coenzyme in carboxylation reactions (C0 2 carrier)
coenzyme-like carrier of hexose groups
- protein kinase A (cAMP-dep.) - serine/threonine A-kinases
protein kinase B (AKT)
- protein kinase C (Ca2+-dep.)
- binds to cytoplasmic face of PM
- stim'd by Ca2+ & diacylglycerol (DAG)
- atypical PKCs - not activated by Ca2+ & DAG (diacylglycerol)
synthesis
Precursors AA Details
a - k e t o g l u h ~ + ~ glutamate amination
PKG - protein kinase G (cGMP-dep.)
PKR - protein kinase R (dsRNA-dep.)
AMPK AMP-activated protein kinase
metabolic master switch regulating glucose uptake,
oxidation, biogenesis of GLUT4 and mitochondria
Vitamins \ .t' 1 tit uw-\.oAI i\ I >'VVSVitamin B6 {Pyndoxal phosphate (PLP))
cofactor for aminotransferase b/w glutamate & other AA
Vitamin B 2 (cobalamin) - transfer methyl groups (methionine syn
Fat solubleVitamin A (retinal)
regulates gene expo in epithelial tiss., rod & cone response
. retinoic acid activates hox genes
Vitamin D3 (cholecalciferol) - converted to 1,25-
dihydroxycholecalciferol, regulates Ca2+ uptake & maintenance
Vitamin E (tocopherols) - destroys oxygen radicals
Vitamin K
essential for prothrombin production
essential for glutamate y-carboxylation
Vitamin Coenzyme Reaction
Thiamine (B1) Thiamine Transfer of aldehydpyrophosphate
Riboflavin (B 2) FADH Redox
I Niacin NADH/NADPH Redox
Pantothenic acid Coenzyme A Transfer of acyl gro
Pyridoxine{Bb Pyridoxal phosphate-¥l{ Amino acid activatio
Biotin (H/B7) Biotin Transfer of CO2
Lipoic acid Upoamide Redox, acyl activatio
Folic acid Tetrahydrofolate Transfer carbon gro
Cobalamin I Transfer methyl gro
MINO ACIDS PROTEINS
AA characteristics
ionization
in solution, AAs act as zwitterions (dipolar ions)
isoelectric point pI) - pH where molecule has no net charge
Enzyme/lnterm Cof
glutamate dehydrogenase PLP
(glutamate)#glutamine reg'ed by adenylation glutamine synthetase
#proline, #arginine
oxaloacetate aspartate transamination PLP
(aspa rtate) asparagine, *threonine, *Iysine a s p a r t y l - ~ - A M P + glutamine 7 asparagine
rtate + cysteine *methionine
#serine
#glycine serine hydroxymethyltransferase H4folate
(serine)#cysteine , , , , , + If id, ~ I
serine + methionine ( cystathionine adoMet
alanine-- transamination by PLPpyruvate
*valine, *Ieucine, *isoleucine • valine leucine - 2
PEP *phenylalanine
+ tyrosine chorismate - branch point phenylalanine hydroxylase
*tryptophani
PRPP *histidine i adenine + riboseI
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pyridoxal phosphate PLP/Vitamin B6)
o aminotransferase b/w glutamate & other AAs
tetrahydrofolate HJolate) - transfers 1 carbons
o dihydrofolate reductase -dihydrofolate-7tetrahydrofolate
S-adenosyl methionine adoMet) - transfers methyls
starvation
stringent response regulation coordinated w/ [AA]
uncharged tRNA bind ribosomes
stringent actor binds ribosome
stringent factor catalyzes formation of ppGpp & pppGppppGpp binds rRN\ reducing synth
9 \l\. >
degradation
s converted to glucose & lipids
amino transferred to a-ketoglutarate (glutamate)
branched-chain a-keto acid dehydrogenase complex
o in extrahepatic tissues (muscle, adipose, kidney, & brain)
o unbranches leucine, isoleucine & valine
non-branched-chain amino acids degraded in liver
o aminotransferase that converts branched-chain amino
acids to a-keto acids absent in liver
Degraded Ipyruvate tryptophan, threonine , alanine, serine, cysteine, glycine I
acetyl-CoA phenylalanine, tyrosine, tryptophan, isoleucine, threonine i
(leucine, lysine (only ketogerifcf - ketogenic)
-ketogluta rate5 carbon skeletons) proline, glutamate, glu tamine
(converted to 5 carbons) arginine, histidine
Fumarate phenylalanine, tyrosine
succinyl-CoA threonine, methionine,d:s01eucine, val in?)
Oxaloacetate aspartate, aspargine
non-protein biomolecules
nitrogen donors in variety of biosynthetic reactionsy-aminobutyrate (GABA)
porphyrins (4 porphobilinogens)
+ arginine & methionine
o phosphocreatine - energy buffer in skeletal muscle
+ glutamate & cysteine
o GSH glutathione) - redox buffer
cotecholamines - dopamine, norepinephrine, epinephrine
melanin (tyrosine 3-monooxygenase (tyrosinase))
+ phenylalanine
o lignin (cell walls of plants)
auxin - plant growth hormone
serotonin
niacin (vitamin in NAD/NADP)
nitric oxide (NO)
histamine (decarboxylated)
Types
single secondary structure (support, shape, protection)
multiple secondary structure (enzyme & regulatory)
- stable, partiall y folded protein state
I
I
I
protein disulfide isomerase POI) - shuffles disulfide bonds
peptide prolyl cis-trans isomerase PPI) - converts cis & trans isom
BiP Binding immunoglobulin protein) binds translocated protei
in ER lumen, preventing aggregation or misfolding
Chaperones
Hsp (heat-shock proteins)
o Hsp60 - barrel shaped-chaperonin isolation chamber
o Hsp70 - BiP (in ER) assists folding during synth
ER chaperone proteins - calnexin & colreticulin
heat-shock response - trigged by accum. of misfolded proteins
unfolded protein response - trigged by accum. of misfolded prote
stims increased transcription of ER chaperones
slows protein production
Pr6tein Structure
peptide bonds - usually planar (trans rather than cis config)
Amino acid backbone
o Ca.-N - {J (phi) rotations
o Ca.-C - i /J (psi) rotations
o C-N bond in backbone is rigid
Ramachandran plot - allowed values for {J & i /J
Secondary structures most common aas)a helix (glutamate, metheonine, alanine)
o normally right-handed
sheet (isoleucine, valine, tyrosine)
connect antiparallel Bsheets (glycine, proline, & asparagine
o 8 turn - 4 residue connection
o V urn - 3 residue connection (considerably less commo
Supersecondary structures
8-a-8 loop, a-a corner, a/B barrel (8-22 B sheets)
Targeting signals
N-terminal sequences
targets for chloroplasts, rough ER, mitochon. (usually cleav
Internal sequences
signal patch multiple internal AAs seqs that form 3D signa
nuclear localization signal- targets protein for nucleus
o located almost anywhere not cleaved)
Protein Glycosylation
precursor oligosaccharide - preformed sugar (14) transferred en
Dolichol-lipid molecule that holds precursor oligosacchari
oligosaccharyl transferase - next to translocator Sec61 complex
N-/inked - connected to NHl on asparagines
complex accessible to modifications
high-man nose unmodified - held close to protein
O-Iinked - on OH (serine, threonine, hydroxylysine, hydroxyprolin
[less common]
Transmembrane proteins
hydropathy plots ID membrane-spanning helices (20-30
Ms)
most glycosylated (sugars added in ER & Golgi)
Protein regulation I modification
Modification TargetAA Change
Phosphate Ser, Thr, Tyr Proteins complexing, on/o
Methyl Lysine Histone code
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Acetyl Lysine Histone code
,Ubiquitin Lysine mono - regulates transport,
poly - degradation
Palmityl Cysteine
=associate w/ membrane
for degradation by proteosomes
- proteasome core contains several proteases
triad -- serine, aspartate, histidine
found inside active site of proteases
- binding sites of complexed n z y ~ sPTB - phosphotyrosine-binding
Src homology - SH2 SH3
o SH3 - binds to proline-rich stretches of polypeptide
(TAGs) (3 fatty acids + glycerol)
derived from phosphatidate (diacylglycerol 3-phosphate)
o 1,2-diacylglycerol intermediate
depots of metabolic fuel (lipid droplets, bud off ER)
primary fuel during fasting converted to ketone bodies)proteins - last cellular material to be used as energy
~ 7 S offatty acids from triacylglycerols recycled
holipids / phosphoglycerides (phosphate + glycerol +
phosphatidylcholine (lecithin) - primary phospholipid
o phosphatidic acid + choline choline + ATP -)
• choline-Pi + CTP -) COP-choline
o methylation of phosphatidylethanolamine (in yeast)
phophatidylserine negative charge
o serine + COP-diacylglycerol
o derived from phosphatidylethanolamine (in mammals)
phosphatidylethanolamine
o (decarboxylation of phosphatidylserine)
important phospholipid in myelin heart tissue
glycerophospholipids w/ acyl chain in place of glycerol
sphingosine instead of glycerol
sites of biological recognition (define human blood groups)
ceramide - basic structural unit of sphingolipids
o fatty acid - amide linkage - sphingosine
phosphatidylcholine + ceramide
only non-glycerol membrane phospholipid
protect membrane
important for cell recognition/adhesion
o cerebrosides myelin (galactose reSidue)
o gangliosides - contain NANA (sialic acids) negative charged
o galactosides neutral charge?
- predominant membrane lipid in plant cells
Fatty acid synthesis
Location cytosol of higher eukaryotes, chloroplasts in plants
Details
CO2 or HCO r ) needed for first step
acetyl groups transferred across mito.memb. as citrate
NADPH used from citrate leaving mito pentose i pathway
fatty acyl grows by 2C from malonate (C02 lossed each step)
final product palmitate (16 C or stearate (18 C)
fatty acid synthetase complex - synth from acetyl-CoA
polypeptide w/ 6 enzyme activities + acyl carrier protein AC
. acetyl-CoA + CO2 - malonyl-ACP + acetyl -) acetoacetyl-ACP + CO
acetyl-CoA carboxylase - rate-limiting step
acetyl-CoA + CO 2 - malonyl-CoA
malonyl-CoA-ACP transferase (MT) - malonyl-CoA -) acetoacetyl-A
Squalene
3 0 ~ C l i n e a r precursor of cholesterol (6 isoprene units)
Synthesis
(1) 3 acetates -) mevalonate (6-C)
(2) mevalonate -) activated isoprenes(3) polymerization of six S-C isoprenes to squalene
HMG-CoA reductase - rate-limiting enzyme in mevalonate pathw
Cholesterol
steroid nucleus - 4 fused rings; 3 w/6-carbons 5-carbon
principle sterol (alcohol w/ OH at C-3) in mammals
lesser amounts in plants fungi (not in chloroplast memb)
OH of cholesterol steroids from O2
decreases permeability
high concentrations in lipid raft, endocytic membranes
Cholesterol Synthesis
(4) 3 isopentenyl pyrophosphate (5-carbon) condense to 15 car
(S) 2 is-carbon combine to 30-carbon molecule
(6) 30 carbon cyclized to form cholesterol
Major synthesis sites - ER of liver small intestine
Biomolecules derived from Cholesterol
vitamin D (essential control of calcium metabolism)
bile acids - aid in digestion of lipids (conjugated by glycine)
steroid hormones
glucocorticoids, mineralocorticoids, sex hormones
Fatty Acid Cholesterol TransportStructure
apolipoproteins, phospholipids, triacylglycerols cholestero
chylomicrons
carry triacyiglycerols from intestine to adipocytes for storag
VWL very low density lipop.)
carry cholesterol esters, triacylglycerols (from liver to tissue
LOL low density /ipop.)
lipoprotein lipase degrades TAGs, converting VLDL to LDL
transports cholesterol throughout body
taken up by endocytosis B-l00 apolipoprotein)
HDL high density lipop.)
removes cholesterol from blood, carrying i t to liver
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degradation
-7 D-glyceraldehyde 3-phosphate -7 glycolysis
generate acetyl-CoA from fatty acids
animals cannot convert fatty acids into glucose b/c degradation
thru CCA does not lead to increase in oxaloacetate
a oxidation - in peroxisomes when ~ c a r b o n is methylated
oxidation - in mito. matrix perox., cyclegen 2 I\lADH,
w oxidation - in liver/kidney ER, cycle gen 1 NADH 1 FADH2
epimers - sugars that differ in config around 1 carbon only
anomers - isomeric form that differ at anomeric carbon
- a-isomer - OH in axial position (down)
- ~ i s o m e r - OH in equatorial position (up)
mutarotation - interconvertible thru noncyclic intermediates
reducing end - end w/ free anomeric carbon w/o glycosidic bond)
nonreducing - sugar w/ O-glycosidic bond on anomeric carbon
aldose - carbonyl group at end of chain (forming an aldehyde grou
- hemiacetal cyclic form of aldose
- aldonic acids - oxidation of carbonyl/anomeric carbon- uronic acids - oxidation of C6 carbon
Breaks carbon bond carbon - 2nd
C from carboxyl C-
3)]
ketose - carbonyl group internal
perox. mito. in mammals, only peroxisomes in yeast/plants
malonyl-CoA shuts down ~ o x i d a t i o no prevents futi le cycle (simul. fatty acid synth
oxidation)
enoyl-CoA isomerase - removes cis double bonds
u nsatu rated)
gens 1 NADH, 1 FADH 2 1 acetyl-CoA per cycle
odd-number fatty acids - propionyl-CoA
-7 methylmatonyl-CoA -7 succinyl-CoA -7 CCA
acyl-CoA dehydrogenase
o removes H a C, forming trans double bond
enoyl-CoA hydratase
o adds H20 across double bond
3-L-hydroxyacyl-CoA dehydrogenase
o converts hydroxyl to keto
acyl-CoA acetyltransferase (B-ketothiolase)
o thiol group cleavage by CoA
shuttle
carnintine transports fatty acyls <::14 C thru mito. inner
memb.
acyl-CoA -> fatty acyl-carnitine -> fatty acyl-CoA
long fatty acyl-CoA synthetase
o CoA + fatty acid -7 fatty acyl-CoA
carnitine acyltransferase I
carnitine-acylcarnitine translocase
o (acyl-carnitine/carnitine transporter )
carnitine acyltransferase
carbon - carbonyl carbon in carbohydrate ring structure
- mirror image stereoisomers
- non-mirror image stereo somers
substrate product Enzyme
glucose glucose 6-phosphatehexokinase
ATP ADP
G6P fructose 6-phosphate phosphoglucose isomerase
F6P fructose l,6-bisphosphatephosphofructokinase-l
ATP ADP
- hemiketal cyclic form of ketose
pyianoses - 6 membered carbon ring
furanoses - 5 membered carbon ring
Naming 5 -6
2 0 : 4 ~ 5 , 8 , 1 1 , 1 4 ) - 20 Carbon chain, double bonds at 8-9, 11-12, 14-1
Non-Glucose Sugars
lactose -7glucose -7 glucose 6-phosphate
galactose -7 glucose 1-phosphateglucose -7 glucose 6-phosphate
sucrose -7fructose -7
fructose 6-phosphate or
glyceraldehyde 3-phosphate
man nose -7 fructose -7fructose 6-phosphate or
glyceraldehyde 3-phosphat
trehalose -7 glucose -7 glucose 6-phosphate
dextrans - bacterial + yeast polysaccharides
GLUCOSEP THVV YSGlycolysis/gluconeogenesis regulation (substrate/futile cycle)
- fructose 6-phosphate <-(FBPase 1) / (PFK-1)-> fructose 1
bisphosphate
fructose 2,6-bisphosphate F2,6BP)
- regs glycolysis gluconeogenesis thru PFK-1
- (activates glycolysis) FBPase-1 (inhibits gluconeogenesis)
- made/broken down by PFK-2/FBPase-2
0 protein w/ 2 enzymatic functions
0 regulated by glucagon insulin controls thru cAMP
Inhibits ActivatesEnzyme
Directly Indirectly Directly Indirectly
PFK-l ATP, citrate F2,6BP Pi
FBPase-l(l,6) F2,6BP AMP triose p h o s p hPFK-2 triose phos phates Pi insulin
FBPase-2 2,6) F6P insulin
Inhib Stim irrev
G6P NADP/NADPH? pentose Pyes
p ,f:i1lA C
ATP, citrate ADP/AMP, F2,6PYes
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~ dihydroxyacetone phosphatealdolase
glyceraldehyde 3-phosphate
DHAP GADP/G3P triose phosphate isomerase
GADP/G3P 1,3-bisphosphoglycerateGADP/G3P dehydrogenase
. NAD+ + Pi NADH
1,3BPG 3-phosphoglyceratephosphoglycerate kinase
ADP AlP
3PG 2-phosphoglycerate phosphoglycerate mutase
2PG phosphoenolpyruvate enolase
PEP pyruvate
ADP AlPpyruvate kinase
\
mainly from AAs from degraded proteins
primarily in liver (smaller extent in renal cortex)
2 pyruvate -> glucose (consumes 4 ATP, 2 GTP, & 2NADH)
bypass irreversible steps (1,3, & 10)
1 t
high nrg compound
ATP, alanine, acetyl-CoA,
long-chain fatty acidsyes
glucose 6-phosphatase removes Pi from G6P
o in ER of kidney & liver only
o compartmentalized to prevent abortion of glycolysis
Glycogenesis (glucose glycogen)
1 glucose + ATP glucose 6-phosphate + ADP
G6P -(glucose 6 - p h o s p h a t a s e ) ~ glucose + Pi
[glucose-1-phosphate + UTP UDP-glucose + pyrophosphate]
glycogen al-4Iink, al-6 branches every 8-12 residues
F6P + ATP fructose 1,6-bisphosphate + ADP
2: F1,6BP -(FBPase-1H F6P + Pi
phosphoenolpyruvate + ADP -> pyruvate + ATP
1: OXAc -(malate d e h y d r o g e n a s e ) ~ malate OXAc
allows transported out of mito.
GTP -(PEP c a r b o x y k i n a s e ) ~ PEP + C02 + GDP
glucose level
Lowers - insulin Ci+ channels triggers release from pancreas)
o glucose uptake, glycolysis, synth of fatty acids
o inhibits fatty acid mobilizati on (glycogen & TAGs storaged)
Raises - epinephrine glucagon cortisol
o gluconeogenesis in liver (inhibit glycolysis)
o glycolysis in muscle
o mobilizes fatty acids from triacylglycerols in adipose
transporters
UT4 - adipose & muscle, sequestered in absence of insulin
-liver, pancreas, hypothalamus, intestine (reabsorbs glucose)
glycogen phosphorylase phosphorylates nonreducing sugar
until 4 sugars upstream from branch
o glycogen phosphorylase alb a-active b-Iess
o controlled by pp-tion by phosphorylase kinase
o stim AMP
o inhib: ATP, glucose (binds to phosphorylase a b)
debranching enzyme
o transferase trans 3 sugars prior to branch to main strand
o glucosidase hydrolyzes branched glucose
phosphoglucomutase G1P glucose 6-phosphate
Enzyme
glycogen synthase lb (active /inactive)
o adds glucose to glycogen (requires 8 glucose chain)
glycogenin synth glucose primer for glycogen synthase I
o inhib by glycogen synthase kinase GSK-3) [3x pp d] rrprimed w i 1 pp by casein kinase II (CKII)
EnzymeInhibits Activates
Directly Indirectly Directly Indirect
phosphoprotein insulin
phosphatase 1
protein kinase A cAMP epin.
protein kinase B insulin
phosphorylase PP-1 PKA Ca +
kinase a/b
glycogen PP-1.r AlP n' - i phosphorylase peptin, ep
phosphorylase a/b kinase, AMP2+
glycogen synthase PKB insulin
kinase 3
glycogen synthase GSK-3 epin., C? PP-1, casein
kinase 2
CITRIC CID CYCLE Krebs)Pyruvate dehydrogenase complex - pyruvate N A D + - N A D H ) ~ C
+ acetyl-CoA
3 enzymes - 5 cofactors
o (24) Pyruvate dehydrogenase -thiamine
pyrophosphate TPP)
o (24) Dihydroli poyl transacetylase -lipoate & CoA
o (12) Dihydroli poyl dehydrogenase - FAD & NAD
Regulation
o Inhibited by: ATP, acetyl-CoA, NADH, fatty acids
o Stimu lated by: AMP, CoA, NAD+, Ca2
+
Citric acid cycle acetyl-CoA 3NADH + 3H+ + 2C0 2 + FADH2 + GT
substrate product notes
1 citrate synthaseacetyl CoA citrate
oxaloacetate + H2O
2 aconitase citrate isocitrate
isocitrate dehyd rogenaseisocitrate a-ketoglutarate
3NAO+ NAOH + H+, CO 2
4 a-ketoglutarate dehydrogenase complexa-ketoglutarate succinyl-CoA heme
NAO+ NAOH + H+, CO 2
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5 succinyl-CoA synthetasesuccinyl-CoA succinate
6 succinate dehydrogenase
7 fumarase
GOP
succinate
Q(FAO)
GTP
fumarate
fumarate malate f- urea cycle
~ 8 ~ ~ ____ ~ a ~ l a ~ t e ~ d ~ e h ~ y ~ d ~ r ~ ~ g e ~ n ~ a ~ s ~ e ______ L m r a ~ l ~ a t ~ e ~ N ~ ~ f V ~ __ o ~ x ~ a ~ l o ~ a ~ c ~ e t ~ a ~ t e ~ ~ N ~ A ~ O ~ ~ ~ _________
c reactions - cofactor - thiamine pyrophosphate
replenish CCA intermediates
pyruvate carboxylase- pyruvate + CO2 o ~ a l o a c e t a t eo biotin - prosthetic group - COf-carrier
o important to gluconeogenesis CCA
o stim acetyl-CoA
ketone bodies - accumulate when gluconeogenesis in liver
depletes oxaloacetate (acetyl-CoA oxidized into ketones)
o synth in liver mitochondria
o acetone, acetoacetate, D-6-hydroxybutyrate (feeds brain)
o converted back to acetyl-CoA in brain, muscles for fuel
of Citric Acid Cycle
Enzyme Inhibits Stimulates
ATP, acetyl-CoA, AMP, CoA,
NADH, fatty acids NAD+, ci+
citrate synthaseATP, citrate, NADH,
ADPsuccinyl-CoA
dehydrogenase ATP ADP,ci+
a-ketogluta rateNADH, succinyl-CoA ci+
e cycle (modified CCA in plants, microorganisms, etc.)
allows synth of sugar from fat (acetyl groups to succinate)
step 1-3, ident ical to CCA (acetate -> citrate -> isocitrate)
o bypasses steps in which carbon is lost as COz
isocitrate dehydrogenase
o pp'd > glyoxylate cycle
o depp'd -> citrate acid cyclestep 4 isocitrate -(isocitrate I y a s e ~ succinate + glyoxylate
o succinate CCA
o glyoxylate + acetyl CoA - malate y n t h a s e ~ malate
CCA / gluconeogenesis
used to make nucleotides coenzymes
o RNA, DNA, ATP, NADH, FADH 2 CoAl
controlled by NADP+ availability
NADPH produced used in anabolic reactions (fatty acid synth)
+ 2 NADP+ + H20 -> ribose 5-phosphate + CO 2 + NADPH + 2H+
phase
Reactants Product I Enzyme
IG6P + NADP+ 6-phosphoglucon I glucose 6-phosphateI • dehydrogenase
+ H20 6 P h O ~ 6-phosphoglucolactonase
6PG + NADP+ ribulose 5-p 6-phosphogluconate
NADPH +CO 2 dehydrogenase
R5P ribose 5-phosphate I phosphopentose isomerase
I Acceptor Regeneration phase
sugar phosphates 56-carbon phosphates
NU LEOTIDES NITROGEN Y LE
Nucleotide Precursors
- AAs, ribose 5-phosphate, CO 2, NH3
- N for purines from glutamine, glycine, aspartate
- aspartate precursor of pyrimidines
ribonucleotide reductase - ribose to deoxyribose NDPs
adenylate kinase - 2 ADP _ M g 2 + ) ~ ATP + AMP
de novo pathway
- pyrimidine bases assembled prior to attachment to ribose
o carbamoyl phosphate + aspartate + ribose 5-phosphate
o 5-phosphoribosyl-l-pyrophosphate PRPP)
• initial acceptor of pyrimidine ring framework
o orotate - a pyrimidine ring framework + riboseo UMP UTP CTP (cytidylate synthetase)
o dUMP + N5,N
1o-methylene-THF ~ dTMP
- purine bases assembled on ribose
o ring built from 5-phosphoribosylamine (committed step
o A G derived from inosine monophosphate IMP)
o coenzyme: tetrahydrofolate
o IMP adenylosuccinate AMP
o IMP xanthylate (XMP) GMP
salvage pathway
recycled base + 5-phosphoribosyl-1-pyrophosphate PRPP) NM
Nitrogen cycle
- bacterial fixation - Nz NH3
o 16 ATP per N2 fixed (2 ATP per electron pair added)
o symbiotic bacteria in roots of leguminous plants
o access to large energy reservoir of plant
- soil bacteria - NH3 N0 3-
o plants - N03- NH3
- all organisms - NH3 AAs nucleotides
- denitrifying soil bacteria - N0 3- Nz
Nitrogen transport
- glutamate - intracellular ammonia carrier
- glutamine alanine - extracellular ammonia carriero excess transported to liver Urea cycle)
Transaminases
- glutamine synthetase
o main regulatory enzyme in nitrogen metabolism
o glutamate + ATP + NH3 glutamine + ADP + phosphate
- glutaminase - glutamine ~ glutamate + NH/
- glutamate dehydrogenase
o glutamate ~ a-ketoglutarate + NH/
Urea cycle
- birds reptiles excrete uric acid (uricotelic animals)
- takes place in liver mitochondria cytosol
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NH4' -7 carbamoyl phosphate -7 urea
1 N from NH/, 1 N from aspartate
Reactants Products Enzyme
2ATP+ carbamoyl phosphate carbamoyl phosphate
HCO g + NH/ 2 ADP + i synthetase I (mito.)
carbamoylornithine
phosphate + citrulline + Pi
ornithinetranscarbamylase (mito.)
citru lline + argininosuccinate argininosuccinate
aspartate + ATP +AMP+PP; synthetase
argininosuccinate arginine + fumarate argininosuccinate lyase
arginine + H2O ornithine + urea arginase
REPLICATION- temperature at which half of DNA helices melt apart
DN absorbs more light than annealed
forms
B form - right-handed, Watson-Crick structure
A form - more compact (wider, shorter) than WC
Z form -left-handed, longer + narrower
tein pairing - DNA that allows for trip lex DNA strands
4 strand binding between guanosine
A - mirroring leads to triple helix
- inverted DNA repeat - form hairpins or cruciform
2-fold symmetry across 2 strands of DNA
can help dislodge/unbind histones
plectonemic interwined in simple and regular way
o extended r i g h t ~ h n d e d coils
solenoidal tight left-handed coiling
# circular DNA strand wound about another
(LK Tw + Wr, Tw = wist #, Tw =writhe number)
-linked DNA strands (like links in a chain)
- Type I breaks one strand, retains energy to reseal (no ATP)
o DNA gyrase negatively supercoils (underwinds) DNA
- Type II - breaks both strands
- DNA is inverted causing change in gene expression
type locus - changeable sequence where mating type stored
ng - fusion of two haploid cells
& cro repressor proteins - repress other's synth
prophage & lytic states
largely consist of short, repeated DNA sequences alpha
satellite DN
over hundreds of thousands of bps
do not seem to contain centromere-specific DNA sequences
defined by protein assembly rather than specific DNA seq
island
CG least frequent dinucleotide
o large % near of promoters
• methylat ion may inhibit expression
CG suppression -loss of Cs due to methyl + deamin (C-7T)
Genome
LINEs - long interspersed nuclear elements (>Skb)
retrotransposons that lack LTRs (long terminal repeat)
SINEs short interspersed nuclear elements «500
Retroviral-Iike elements
DNA-only transposoh 'fossils'
SSRs (single-sequence repeats/highly repet itive sequences)
generally less than 10bp repeated up to a million times
satell ite DNA - unusual base comp causes it to migrate as
satellite bands (separated from rest of DNA)
Transposons (mobile genetic elements)
produce many genetic variants necessary for evolution
Complexity
insertion sequences simple)
o seq required for transposition & transposase genes
complex transposons - contain one or more genes in additio
to those needed for transposition
Types
DNA-only transposons responsible for antibiotic resistance
o Bacteria contain mostly DNA-only (few nonretrov iral
retrotransposons)
o exist only as DNA move by cut-and-paste transposi tion
replicative transposition
retroviral-Iike retrotransposons
o mRNA -7 reverse transcriptase -7 RNA/DNA -7 DNA/DN
-7 integrated by intergrase
o require RNA polymerase & reverse transcriptase
nonretrovira l retrotransposons
o responsible for repeated DNA seq
o require endonuclease + reverse transcriptase complex
movement
o direct transposition - movement from one site to anoth
o replicative transposition - replication into new site
Transposon-related terms
transposase protein that promotes transpositions
intergrase - viral transposase
cointegrate donor & target DNA intermediate during replicative
transposition
Genetic Variation
SNPs (single-nucleotide polymorphisms)
extensively characterized i n t r ~ s p e c i e s gene variations
Genes
haplotype blocks - sets of alleles that are inherited in clusters
complex traits - do not follow simple inheritance (often polygenic
site-directed mutagenesis - intential mutation to determine funct
transgenes - foreign or modified genes
synteny same genes in the same order across species
pseudogene - duplicated gene deactivated by mutations
X-inactivation - occurs after Nl000 cells formed (mosaic)
x-inactivation center - XIST RNA
10% of genes remain active
unique histone composition/modification pattern
Barr body - condensed inactivated x-chromosome
dosage compensation
o inhibition, up regulation, down regulation
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heterochromatin - highly condensed, inactive
euchromatin less condensed, active (acetylated histones) ,
o hypersensitive sites
• actively transcribed regions sensitive to DNase I
AT sequences more compressible
position effect - gene silencing of positions win or nearby
heterochromatin
chromatin jiber - 30nm, zigzag model of packing
metacentric - middle centromere
submetacentric - slightly off-center centromere
acrocentric - more off-center
telocentric centromere at or near end
isochromosome -lost arm replaced by duplicate of other
highly conserved, rich in lysine arginine +)
structure: fold - 3 helices + 2 loops
N-term tail covalent modification determines chromatin
o Acetylation/methylation of Iysines
o Phosphorylation of serines
o H4 tai l- important for nucleosome-nucleosome linking
C-term tai l- mono or polyubiquitinated
variant core histones
o expressed at different times during development
o CENP-A - centromere-specific H3 histone
Hl-l inker histone (larger less conserved)
DNA sequences - protect spread of histone code
HS4 sequence - protects l3-globin expression
20 sets of histone genes
synth mainly in Sphase (1't ranscription, -J.,degradation)
HATs histone acetyltransjerases)
o neutralize +) charge, destabilizing chromatin
o (type A - nuclear, type B - cytosolic)
HDACs histone deacetylases)
o compacts chromatin, attracts other proteins
histone chaperones chromatin assembly factors
SW1/SNFNURF
- remodel chromatinSMC proteins structural maintenance of chromosomes
different combinations of tail modification important to
o marking
• replicated chromatin
• DNA damage
o Inactivation
Heterochromatin formation
Hox silencing (developmental gene silencing)
• X chromosome inactivation
Code-reader complex - adapter that interprets histone code
recruits protein complexes
nucleosome
basic unit of chromosomal organization, N200 bps
o linker DNA - up to 80bp
o nuclesome core
histone octamer 2x H2A, H2B, H3, H4)
• dimers H3+H4, H2A+H2B) tetramers (2x H3+H4)
• 147bp DNA (1.65 turns), left-hand coiled
location - depends on seq, presence of other bound protein
nucleosome sliding - movement along DNA catalyzed by
remodeling complexes
polytene chromosome
bands (dark) - inactive
interbands (light) - active
puffs (Balbiani rings) - transcriptionally active DNA
DNA binding motif s
helix-tum-helix
o regulatory proteins -lac trp, CAP
o homeodomain - developmental
zincjingers hold together by zinc
o steroid receptors
leucine zipper moti f 2 alpha helixes - dimer
o transcriptional activators
helix-loop-helix HLH) short a-helix + loop + long a-helix
P-Ioop
Damage/Mutation
Damage
deamination of bases loss of amine)
o C 7 U - 100 per day per cell
loss of bases - bases dissociate from ribose (N-glycosyllinkag
o depurinat ion 5000 per cell per day
o depyrimidation - 500 per cell per day
UV induced pyrimidine dimerso result in deletion or substitution of 1+ base pairs
oxidative damage occurs to double bonds C-C bonds
hydrolytic attack - N-C bonds, phosphate
uncontrolled methylation - nitrogen, flipping out
radiation - breaks purine rings phosphodiester bonds
Tautomeric shifts
cause G-T A-C binding
G T - keto 7 enol
A amino 7 imino
Mutation rate
1 in 10
nucleotides
20,000 DNA alterations per cell per day
Mutations
point (single site} - single nucleotide or very small part
o transition one purine (or pyrimidine) to other
o transversion purine to pyrimidine vice versa
o nonsense mutation - results in premature termination
o missense mutation different translated M
insertion / deletion loss of or gain of nucleotide(s)
o jrameshift insertion/deletion altersing reading frame
inversion - segment is reversed
translocation - segment migrates from one chromo to anoth
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germ-line - allows mutation to be passed to progeny
polar- mutation that affects subsequent genes (polycistronic)
Mutations
conditional phenotypic change under cert. conditions (temp)
loss offunction reduced gene act ivity (usually recessive)
o null complete loss of gene activity
gain of unction - increased or inappropriate activity (usually
dominant)
o dominant-negative - mutant interfereswI normal gene
suppressor- counteracts phenotypic effect of prior mutation
o back mutation reversion to previous sequence ' 'o intragenic - with in 1 gene
o extragenic - within 2 genes
cal repair
photolyases - use light energy to reverse dimmers
- depends on methylation of A resides (in GATC seq)
sion repair
excision repair
DNA glycosylase exinuclease)o removes base from DNA lesions (creates AP site)
AP endonuclease
o cuts phosphodiester at AP (apurine/apyrimidine site)
cleaves phosphodiester backbone on both sides of distortion
DNA around site of damage is excised & resynthesized
TFIIH - opens up excised DNA
repair more efficient wIn actively transcribed genes
RNA polymerase stalls on lesions & directs repair machinery
ologous end-joining ends ligated wI loss of 1 nucleotides
requires long homologous segments
exonuclease 5' end, strand invasion, branch migration
genetic exchange between DNA wI extensive homology
occurs wI highest frequency during meiosis prophase I
resolution by crossover or non-crossover (gene conversion)
can repair DNA damage & restart replication forks (primosome)
too much (loss of heterozygosity)
too little (mutations lead to cancer)
- integrates & excises during recombination
can result in inversion, turn ing genes on or off
lliday intermediate - X orm
ladderlike structure blw homo. chromosomes during meiosis
interconnected by transverse filaments TFs)
- individual crossover events blw nonsister chromatids
only visible during diplotene
ensures at least one crossover blw homologs
crossover inhibits formation of others
of Replication)
rich in AT (weaker hydrogen bonding)
binding site for aRC, aRC recruitment proteins
10,000 in humans excess ensures replication pr ior to divisio
o 30,000-250,000 np bIn replication origins in mammals
replication unit - origin cluster activated together (20-80)
o activate in reproducible order
ARSs autonomously replicating sequence)
o Euk. chromosomal origin of replication
ORC Origin Recognition Complex)
remains associated wI origin through cell cycle
pre-RC pre replication complex)
aRC, DNA, Cdc6 & Cdt l helicase loading proteins
form only during Gl phase Cdk activity low)
2x Cdc6 + 2x Cdtl recruit 2x Mcm - helicase 6 subunits)
geminin - binds & inhibits Cdtl
disassembled during 5 phase (1'Cdk, Mcm pp'd starts repl.)
l 5 & M-Cdk & .J APC/C activity blocks pre-RC formation
primosome DnaB helicase + DnaG primase (lagging strand)
replisome - entire replication complex at replication fork
DNA replicationIn prokaryotes:
DnaA binds 4x 9-bp
HU denatures DNA in 3x 13-bp repeats
helicase further unwinds DNA
SSB proteins bind RPA in eukaryotes)
E. coli DNA polymerases
DNA polymerase I
removes RNA primers
Klenow fragment DNA poll wI removed 5'->3' exonuclea
DNA polymerase III
more processive than DNA poll blc of subunits
a subunit - polymerization activitysubunits - DNA clamp, prevents dissociation
E subunit - 3' to 5' exonuclease activity
T subunits links 2 core polymerases & coordinate synthes
Mammalian DNA polymerases
DNA polymerase a - synth primers & start of Okazaki fragments
DNA polymerase - base excision repair
DNA polymerase 6 - primary pol for both strands
DNA polymerase V - mitochondrial DNA
Viral DNA polymerases
DNA viral genomes encode own DNA polymerase
Mitochondrial & plastid DNA - circular, 2 ori, multiple copies (2-1
RN TR NSCRPTIONtranscriptome - all transcribed genes in an organism mRNA + rRN
RNAs
snRNAs (small nuclear) splice pre-mRNA
snoRNAs (small nucleolar) - modify rRNAs
many encoded in introns of ribosomal protein genes
scaRNAs (small cajal) modify snRNAs + snoRNAs
miRNAs & stRNAs (micro & small temporal )
complementary to 3' UTR of mRNAs
block translation & target mRNA for degradation
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initiates RISC (RNA-induced silencing complex) formation
As - small interfering compact chromatin & degrade mRNA
- telomere synthesis,x-chromosome inactivation
& ribosomal proteins
200 rRNA gene copies per haploid genome in humans
precursor
~ 1 0 0 methylations (2'-OH positions)
~ 1 0 0 isomerizations (uridine -> pseudouridine)
modified & cleaved by snoRNAs\
also contain genes for DNA primase, RNA polymerase, EFs
r-protein in each operon functions as repressor
o repression if r-proteins exceeds rRNA
RNase 1/1- trims pre-rRNA
heavily modified (over 50 types of modification)
some eukaryotic tRNAs have introns
Structure
o amino acid arm
CCA 3' (added by tRNA nucleotidyl transferase),
• AAattached to either 2 or 3 hydroxyl
• RNase P - cleaves 5 of tRNA
• RNase D - trims 3 of tRNA
o anticodon arm
o D arm & TtjJC arm
- different anti-codon tRNA coding for same AA
synthetases Mg2+ dependent, 2 steps, ATP
- inosine/inosinate in 3 position base-pairs w/ U, C, or A
5' wobble - GUG may initiate translation in prok. ( ~ 1 4 )o UUG & CUG even less frequent at start codon in prok.
moncistronic - code for single polypeptide (most all euks)
polycistronic - code for multiple polypeptides (prok. & viruses)
UTR (untranslated region)
region bIn stop codon & poly-A tail
plays role in mRNA localization & transcrip tion con trols
repressors disrupt comm. bIn tail & cap - slowing translation
carry sites for proteins that accelerate/impede degradation
editing
uracil insertion/deletion
o{gRNA (guide RNA) templates} - frameshifting
A 7 I (inosine) occurs to dsRNA
o ADARs ADR1 liver, ADR2 - bra in, ADR3 - unknown
C-7U
forms RNA-like polymers from RDPs w/o template
Involved in mRNA processing & degradation
Degradation
large protein complex that degrades RNA w/ exonucleases
processing bodies) - cytosolic site of most RNA degradation
RNA Polymerase ,/ Iv.
does not require DNA helicase (ATP)
deacetylates histone tails
limited proofreading (no nuclease activity)
requires template, nucleotides, Mg2+ & Zn2+
pro:
o RNA polymerase holoenzyme
• core enzyme a a ~ w(J subunit/factor
• specificity factor
• binds to promoter
• dissociates from core ns into translation
eu: (3 pols - differential sensitivities to a-amanitin)
o RNA polymerase 1- 5.85, 185, 285 rRNA (45S precursor)
o RNA polymerase II - mRNA
• continues trans, but overshoot lacks cap (degrade
o RNA polymerase 111 tRNA, 55 rRNA, snRNA, other
CTD (C-terminal Domain)
o responsible for initiation & bonding of related complexe
o cap synthesizing complex
• synth 5 cap (5' to 5 7-methylguanosine - protects
eu-mRNA & aids in translation)
o cap binding complex
secures mRNA to RNA polymerase following cappi
Initiation
PIC preinitiation complex) - denatures DNA & helps position RNA
polymerase II
no uniform order of assembly
o (TFIID 7 TFIIB 7 TFIIF 7 TFIIE 7 TFIIH)
consists of 6 general transcription factors
o TFIIA, TFIIB, TFIID, TFIIE, TFIIF, TFIIH
(1) T F I I D ~ T B P (TATA-binding protein) binds TATA box
(2) TFIIB - binds TBP & DNA
3) TFIIA - stabilizes TFIIB-TBP complex (not necessary)
important for nonconsensus promoters which bind TBP wea
(4) TFIIF + Pol II bind TFIIB-TBP complex
(5) TFIIE & TFIIH bind - create closed complex
Transcription initia tion complex - polymerase + transcription fact
TFlIH - unwinds DNA to start transcription ATP)
o Phosphorylates RNA polymerase CTD (C-terminal doma
o Releases polymerase from promoter
Elongation
TFIIE & TFIIH - released in initial 60-70 nucleotides
Transcriptional elongation factors
suppress pausing, decrease likelihood of dissociationcoordinate activites between protein complexes
involved in posttranscriptional processing
dephosphorylated upon termination
p TEFb positive transcription elongation factor b) pp's CTD
Polyadenylation
upstream AAUAAA site
CstF cleavage stim factor) & CPSF cleavage & polyadenylat
specificity factor) transferred from tail to 3' end
PAP polyadenylate polymerase) - adds ~ 2 0 0 A (from ATP)
o slow addition at first followed by rapid addition of 200-
tail acts as timer (mRNA half-life)
o short for bacteria (seconds)
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o varies for euks - long for B-globin (10h), short for growth
factors & regulatory proteins
tail can be extended, extending mRNA life
Poly-A binding proteins - determine final length, help direct
translation
ional controls ( ~ 7 5 of human genes)
transcription attenuation premature termination
o used by many AA synthesis operons
riboswitch secondary structure of mRNA that turns on/off
additional transcription
alternative RNA splicing
quality control
cytosol
5'cap & poly-A tail recognition
EJ (exon junction complex) - stimulates translation
Nonsense-mediated mRNA decay
o stop codon in wrong place causes degradation
ns - exon size much more uniform than intron size ~ 1 5 0 )Introns
Composition: snRNP (RNA + protein) - snRNAsLocation: bound to pp'd tail of RNA polymerase II
SR proteins - mark junctions - recruit snRNPs
U1- 5' splice junction
BBP & U2AF - branch point, mark upstream boundary
U2 - branch point
U4/U6*U5 - triple snRNP - splices
pe splicesome +10 to all U s, recognizes diff nucleotide seq.
splicing RNA - rare - phage, mitochondria, chloroplast genes
Group 1- G attacks, no lariat (guanosine cofactor)
Group II - A attacks, lariat
(rare) - mRNA from diff mRNA spliced together
single 5' exon spliced onto different 3' exons
search for identical site for intron insertion
homing endonuclease copies intron into new homo. gene
retrohoming DNA reverse spliced - spliced intron inserted
by endonuclease reverse transcrip tase
egulation
control region gene promoter & regulatory sequences
polycistronic region translated together from single promotergene + promoter, operator, enhancer, etc
network of operons wi common regulator
HREs (hormone response elements)
o reg. DNA seq binds hormone- receptor complex
operator - bound by repressors & activators
TATA box (Hogness) - 25 bp (bound by TBP)
Pribnow box - -lD bp (binds RNA polymerase subunit)
additional reg. seq. in higher euks bound by activators
can enhance transcript ion of upstream & downstream genes
UASs (upstream activator sequences) in yeasts
UP element (upstream promote r) -40-60 bp
CAAT box - 75-80 bp
initiator start of transcription - +1 bp
terminator sequence that ends transcription
Trans-elements
DNA-binding transactivators bind enhancers/UASs
effector regulates repressor binding
inducers prevents binding of repressor protein to DNA
gratuitous inducer inducer cannot be metabolized
basal transcription factors required at every RNA pol II promote
coactivators/ corepressors do not themselves bind on DNA
mediator mediates activator & transcription machinery
.RNA sequences
Kozak sequence translational start site for ribosome
leaCler - 5 ' end of mRNA preceding initiation codon of first gene
attenuator -lies within leader responsible for attentuation
start codon AUG
stop codons GAA, UGA, UAG
Specific examples
catabolite activator protein (CAP) regs certain prokaryotic genes
complexes wi cAMP, binds promoters
lac operon - transcribedwi
low glucose conc. ([cAMP] high) & higlactose conc. (Lac repressor unbound)
CRE & CRE cAMP response element-binding)
TR NSL TION
Ribosome
small subunit - framework for matching
o pro: 165 rRNA from 305 pre-rRNA
o eu: 185 rRNA from 455 pre-rRNA
large subunit
o pro: 235 & 55 rRNAs from 305 pre-rRNA
305 contains tRNA as intronso eu: 5.85 & 285 rRNAs from 455 pre-rRNA
o catalyzes peptide bond - peptidyl transferase
o Sites:
E - ex it
P - peptidyl-tRNA
A - aminoacyl-tRNA
polyribosomes/polysomes multiple ribosomes translationa
complexed on ER surface
Initiation
Initiation in pro
Translations begins while transcription is still in progress
Initiation complex
o 305, mRNA, fMet_tRNAfMet
, (IF-1, IF-2, IF-3), GTP, Mg2+
305 binds IF-1 + IF-3
o IF-3 prevent premature 305 + 50S binding
mRNA binds 30S-IF-1-IF-3 complex (SD seq pairs wi 165 rRN
fMet_tRNAfMet + IF2-GTP bind AUG
50S subunit binds - GTP hydrolyzed, initiation factors depar
Initiation in Eus
elFs (eukaryotic initiati on factors)
5' & 3' ends tied together - PAB (Poly(A) binding protein)
elF4F complex (eIF4E, elF4G, eIF4A) bindsto 5' cap
o [eIF4A- RNA helicase]
elF4G - binds elF4E & PAB
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elF4F complex - associates w/ elF3 & 405 ribosomal subunit
elongation factors EFs) increase speed & accuracy
binding aminoacyl tRNA (aa-tRNA+EF-Tu-GTP)
o EF-Tu - mediates aminoacyl tRNA entry to ribosome
peptide bond formation (235 rRNA - peptidyl transferase)
translocation (EF-G(translocasel + GTPl [EF-G catalyzes
translocation of tRNA & mRNAJ
o large subunit translocation, then small subunit
translocation
Ts - serves as GEF guanine nucleotide exchange factor) for EF Tu
- initiation on free ribosomes
- binding of 5RP (sig rec. particle) to polypeptide (GTP-used)
- binding of 5RP, polypeptide, & ribosome to 5RP receptor
- dissociation of 5RP from its receptor
- translocation across membrane of ER
- cleavage of signal sequence
translocon -takes signal seq across ER memb
- termination codon in the A site
anslational modifications
In Lumen of ER and/or Golgi
N-term & C-term modifications removed or modified
Loss of signal sequence
proteolytic processing - inactive to active
Phosphorylation 5er/Thr/Tyr
Carboxylation Glu
Glycosylation usually occurs in Golgi
o Asn - N-Iinked oligosaccharides
o 5er/Thr - O-linked oligosaccharides
o isoprenyl groups - Cys - thioether bond
(helps anchor protein in mem.)
o prosthetic groups - biotin, heme
disulfide cross-links
quaternary structure - complexing of subunits
phosphorylation of initiation factors
RNAi - miRNAs, stRNAs bind mRNA (to 3' UTR)
o prevent translation initiation
disrupt elF4E & elF4G interaction
UAA, UAG, UGA
causes binding of water molecule instead of amino acid
interference
dsRNA inhibits genes w/ complementary sequence
can lead to heterchromatin formation
7 2'-S'oligo(A)synthetase 7 RNase L (destroys aU RNA)
7 Protein kinase R 7 apoptosis
high energy phosphate bonds split for each new peptide bond
2 consumed in charging of tRNA w/ AA ATP -> AMP)
2 during synthesis in ribosome
extra energy consumed w/ mistaken connection
Regulation & modification
Translational repressors bind 3' UTR (untranslated region) of mRN
block interaction w/ initiation factors or ribosome
prevent or slow translation
Reading Frame
ORF open-reading frame) lacking termination win codons
translation recoding
proceeding seq. causes codon to be translated differently
translational rameshifting
shift of reading frame as ribosome is translating mRNA
.leaky scanning - ignoring of AUG codon, produces differen t prote
" different or no N-terminal signal sequence
IRES internal ribosome entry site) - bypass need for 5' cap & elF4E
CELLUL R COMPONENTSNucleus
Subnuclear structures
Cajal bodies & GEMS (Gemini of Cajal bodies) - srioRNAs &
snRNAs modify al lItil8g l11fjli i . NS
IGCs (lnterchromatin granule clusters) - stockpiled mature
snRNPs
nucleolus - site ofrRNA transcription
o NOR (nucleolar organizing region) - region containing 4
pre-rRNA genes around which nucleolus forms
Nuclear lamina
protein meshwork, provides support for nuclear membran
disassembles through phosphorylation by Cdk
Nuclear pore complex NPCs)
aqueous, permeable to small molecules «50,000 daltons)
octagonal w/ long filaments
Karyopherins (nuclear transport receptors)o Export
• 5' cap first CBC (cap-binding complex)
• sheds some proteins (nuclear-restricted proteins)
• e e & nuclear export receptor h . ~ c in cytosQL.;
• Ran-GEF in nucleus causes R a n - G t ~ : - - > o o R a i l - G D J yo Import erf .) ({ .
• Ran-GAP in cytosol causes R a f l ' 6 ~ ~• importin bound (via FG-repeats)
• Nuclear import receptors - bind NPC signal
Endoplasmic Reticulum
lumen/cisternal space
cytochrome P450 system
o oxidative metabolism in ER o iver & small intestine
o drug metabolism & detoxification of foreign substanc
sequester ci+ - specific regions of ER specialized for stora
lipid synth - most lipids synth in cytosolic leaf of ER
membrane
o cholesterol, ceramide & fatty acid
protein modification
o glycosylation
o proetolyt ic processing - in ER & post-Golgi
Transport into ER
Most proteins imported during translation
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Signal-recognition particle SRP) - cycles in ER cytosol
Sec61 complex - tetra mer, 1 pore + bind ribosome, proteins
start transfer peptide - binds w SRP - starts translocation
o internal start transfer signal can serve as anchor
stop transfer peptide - anchors protein in membrane
o multipass transmem. proteins - internal start,
alternating stop start seq.
ER resident proteins
o ER retention signal - K[)El (lys-Asp-Glu-leu)
o most complex - too large for most t ransport vesicle's
post-translation translocation requires motor protein
o BiP - pulls in protein
cis - ER side, trans - plasma membrane side
Anterograde transport through the Golgi apparatus occurs
via cisternal progression
catalase urate oxidase (crystallized core)
o c t l se - hydrogen peroxide -> w ter + oxygen
o use O2 to remove H from organic substrates 13
oxidation)
Import
o peroxins - import proteins into peroxisomes
o C-term peroxisome import seq. - Serine-lysine- leucine
o Unknown N-term sequence
osomes - acid hydrolases
hydrolyzes cholesterylesters
o releases cholesterol fatty acids into cytosol
mannose 6-phosphate M6P) - targets proteins for
Iysosomes
osomes - acidic H+ATPase)
early, late recycling - recycles stores membrane proteins
receptors, etc
s (MVBs) - endosome w invaginations
sorting into internal vesicles regulated by ubiquitin
- cytosol
- single-membrane of vacuole
- photosynthetic membrane invaginations
- invagination to which DNA is attached
germ cells PGCs)
cells singled out early on gamete progenitors
development
(sex-determining region of Y) - directs testis development
ls - main suppor t cells in testis
greater quality control than egg development
contribute more mutations (larger number of mitotic divisions)
acrosomal vesicle - secretory vesicle at head of sperm
syncytium - mUltiple nuclei w shared cytoplasm
spermatogonium - rep.)-7 prim. spermatocyte - mei-l)-7 sec.
spermatocyte -(mei -II)- 7 spermatid - spermatozoa (mature sperm
oogonium - rep.)-7 prim. oocyte -(meiOSiS 1)-7 sec. oocyte - mei-
11)-7 oovid - ovum (mature egg)
egg cells
main source of aneuploidy
parthenogenetic - eggs activated in absence of sperm
nurse cells - feed mRNA into oocyte
Fertilization - usually egg is a secondary oocyte
1) capacitation - sperm modif. by female reproductive trac
o changes in glycoproteins, lipids, ion channels RP
o unmasks cell-surface receptors that help sperm bind
o greatly increases flagellum motility
. (2) reach site of fertilization in oviduct
(3) migrate through granulosa ~ l l s surrounding egg
4) bind to cross zona pellucid
(5) bind to fuse w plasma membrane
o fusion activates egg by increasing Ca2+ in cytosol
o sperm donates centrioles (lacking in unfert ilized egg)
polyspermy - more than one sperm fuses
polyspermy blocks
fusion of first sperm causes change in egg plasma membrane
cortical reaction - enzymes change structure of zona pellucid
o second polar body is extruded
aneuploid - cells w abnormal chromosome
euploid - cells w normal chromosome
in vitro fertili zation lVF) - aids infertile women
intracytoplasmic sperm injection lC51) - aids infertile men
Inheritence
genomic imprinting - expression of maternal or paternal gene onl
epigenetic inheritance - inherited protein structure superimposed
on genetic (DNA-based) inheritance
m tern l effect - accumulated mRNA of mother's genes that
dictates early development
OTHER ELL TYPES
Stem Cells
Depend on contact signals from strom l cells
totipotent - any cell type, including extraembryonic
pluripotent - any cell type, but extraembryonic cells (placen
immort l strand hypothesis - stem cells maintain same DNA
transit amplifying cells - divide frequently, leave basal layer
terminal cell differentiation - final specialized characteristics
cellysually permanently stops dividing
apical meristems - undifferentiated pluripotent growing tip in pla
Epithelial cells
keratinocytes - epithelial cells w excessive keratin intermediate
filament synthesis which give epidermis its toughness
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Cells
- construct bone
osteocyte imprisoned in hard matrix
- remove bone (control led by osteoblast signals)
cells
abundant smooth ER (principle site of lipoprotein production)
differentiated cells, no apparent stem cells .
interface between digestive tract and blood
major function - maintain blood glucose level G6Pase) ,
- air-filled sacs of lungs
Type most of wall, thin flat to allow gas exchange
Type I interspersed among Type I, plump, secrete surfactant
antimicrobial peptides that kill p a t h o g ~ n s in mucus layer
most abundant proteins in neutrophils
short (12-50 AAs) positively charged
et cells, ciliated cel/s, macrophages - keep airways cleangoblet cells - secrete mucus
tive cells brush-border cells or enterocytes)
microvilli increase nutrient uptake secrete digestion enzymes
cells
secrete serotonin peptide hormones that act on gut wall
cells - innate immune cells in small intestine
similar to neutrophils, secrete cryptdins (defensin family)
cell niche crypt in gut lining that protects stem cells
acids enzymes
- stims secretion of HCI into stomach, optimal for pepsin
acid - low pH acts as a denaturing agent
- (chief cells) cleaves phe,leu not next to proline}
- cleaves tyr , phe, leu, lie, val, trp, his at high pH
- (duodenum) cleaves lysine arginine not next to proline)
buffering
CO2 + H20 -> H+ + HC03-
cells
l cells - form linings of blood vessels lymphatics
- cell fragments derived from megakaryocytes
(rouget cell, mural cell) .- blood vessel suppor t cells
differentiate into fibroblast, smooth muscle, or macrophage
proteins
n - carries iron in the blood
albumin carries fat ty acids to heart skeletal muscle
lood cell promoting hormones
CSFs)
support hemopoiesis (formation of blood cellular components)
stims
o erythropoietin stims production of erythrocytes
o interleukin-3 lL3) - promotes survival proliferation of
earlier erythroid progenitor cells
lood cell lineage
BFU-E
myeloid stem cell
pluripotent hematopoetic stem cell (hemocytoblasts)
Reticulocyte - immature erythrocyte
Erythrocyte - red blood cell
Hemoglobin
heme - porphyrin ring, synth'd from s J c c i n y / ~ C D AR state - relaxed (higher O2 affinity) / T state - tense (lower
affinity)
O2 bindingo moves heme iron closer to porphryn ring plane
o prevents binding of 2,3-bisphosphoglycerate 2,3-BPG)
lowers O2 affinity
o converts T to R state
H+ CO2 binding
o binding inversely related to O2 stabilizes T form Bohr
effect)
o H+ - binds to several places.
o CO2 - binds a-amino at N-term of each subunit
o transported from cells to lungs kidneys
fetal hemoglobin - higher O2 affinity than mother's adult hemoglo(due to decreased binding of 2,3-BPG)
Hemoglobin S - abnormal form responsible for sickle cell anemia
Mutant ~ s u b u n i t - (negative AA -> hydrophobic AA)
forms fibrous aggregates when O2 released
Oxygen binding models
MWC model/concerted model - all subunits undergo
transformation simultaneously
Sequential model binding of one ligand makes binding of adjace
ligands more likely
Hill plot -log[6/1- 6] v 10g[LJ
Hill coefficient - slope of Hill plot - a measure of cooperativeinteraction between protein subunits
HORMONESpeptides, amine, eicosanoid hormones act at cell surface
steroid, vitamin 0, retinoid, thyroid horomes act on nuclear
receptors
Peptide hormones
leptin
produced by adipose tissue
regulates energy uptake expenditure
acts on arcuate nucleous of hypothalamus - inhibits eating
sympathetic nervous system - uncouples mit ox ph ->
thermogenesis
functions thru Jak/STAT
ghrelin - produced in stomach - producesfeeling of hunger
adiponectin stim fatty acid uptake / oxidation, inhibits fatty acid
synthesis
functions thru AMPK
atriopeptin - atrial natriu retic peptide/ factor AN P AN F)
hormone secreted atria to lower blood pressure
o reduces water , Na+, adipose loads in blood
vasopressin kidneys reabsorb water
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d hormones
- kidneys retain Na+& water
fatty acids
mammals cannot introduce more than 1 double bond into fatfy
acids
obtained from plants
linoleate C-18 cis-t,9, t,12}
a-linolenate (C-18 cis-t,9, t,12,t,lS)
-7 arachidonate (20-C cis-t,s, t,8,t,11,t,14} -7 eicosanoids
glandins, thromboxanes, l e u k o t r i ~ n e s . potent signal
Prostoglandins - regulate synthesis of cAMP (mediate pain,
fever, and inflammation}
Thromboxanes mediates blood Clotting (produced by
platelets)
Leukotrienes powerful signal in aJlergicreactions
- regulate growth/diffe rentiat ion in plants
Plant hormones (growth regulators)
Ethylene - gas molecule
o Fruit ripening, leaf abscission, plant senescence
o Stress signal wounding, infection, flooding
o Stem thickening, shields tip of root, changes growth
direction /ltriple response
auxin - helps plants grow toward light, roots down
- precursors of skeletal muscle fibers
- fuse to repair damaged muscle or allowfor muscle
generally survives for entire animal life time (individualnuclei can be added or lost)
sarcoplasmic reticulum - abundant, modified smooth ER
found in muscle
fast-twitch muscle - fewer mitochondria & less blood supply
than slow-twitch muscle, quicker to fatigue
phosphocreatine / creatine phospha te / phosphagens -
reservoir of high-energy phosphates
myostatin - secreted to limit own growth
nitric oxide relaxes smooth muscle (thru guanylyl cyclase
-7 cGMP -7 protein kinase G}
basic contractile element
sacromere contractile unit
A band- thick filament
I band - only thin filament
M-line - bisects A band (thick filament} where myosin heads
switch polarity (para myosin, C-protein, M-protein)
Z-disk - CapZ & a-actinin - crosslinks & caps +} ends of actin,
bisects I band (desmin}
tropomodu lin - stabilizes minus end of actin
molecular rulers
o nebulin - template protein along length of actin
o titin - links myosin to CapZ
e cycle
1) ATP binds myosin, actin unbinds
(2} ATP hydrolyzed, conformational change & weak binding
F-actin subunit
(3) Pi released, myosin bond strengthened
4} pi release triggers power strokelJ w/ ADP release
Contraction Regulation
tropomyosin- binds actin, blocking myosinattachhlent
troponin T,I,C}
o Toponin 1-T pulls tropomyosin out of normal
binding groove - preventing myosin binding
o Troponin C-binds 1-4 Ca2+causing I to release
actin exposing myosin-binding sites
Neuromuscular transmission
1 nerve impulse causes uptake of Ca2+ (v-gated Ca
2+ chan}
which stim release of acetylcholine
2. acetylcholine binds to nicotinic acetylcholine receptor, in
of Na+ depolarizes muscle
. 3. v-gated Na+ channel lets more Na + in
4. depolarization activates v-gatedCa2+ channels in transve
tubules
5. causes ci gated Ca2+ release channels Ryanodine
receptors) in SR to open - myofibrils in muscle cell contra
Fermentation (regenerates NAD+)
pyruvate -(NADH-NAD+}-7 lactate
pyruvate -> C02 + acetaldehyde -(NADH-NAD+}-7 ethanol
pyruvate -(lactate dehydrogenase}-7 lactate + NAD+
pyruvate -(pyruvate decarboxylase}-7 acetaldehyde
alcohol dehydrogenase-> ethanol + NAD+
o generates molecule of CO 2 from carboxyl
group of pyruvate (carbons 3 & 4 of glucose
acetaldehyde - builds up in liver from ethanol
Cori cycle
when muscles lack sufficient oxygen, pyruvate reduced to
lactatelactate exported to liver
liver converts to pyruvate, then glucose & exported
Glucose-Alanine Cycle
in muscles: pyruvate + ammonia -7 alanine
alaninine transported to liver & converted to glucose & urea
alanine + a-ketoglutarate -7 pyruvate + glutamate -7 glucos
NERVOUS ELLS
neuroblasts - nerve-cell precursors
neurons
- use only glucose & l3-hydroxybutyrate for energy 13
hydroxybutyrate converted to acetyl-CoA via£E1\in brain
-most ATP used for transport of Na+ & K , and maintenance o
potential
glial cells - specialized supporting cells that form myelin
Schwanncells peripheral nerves
Oligodendrites ..:..central nerves
Neuron Ion channels
voltage-gated Na channels responsible for AP (present at Node
of Ranvier along axons)
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K channels - reduce AP firing at levels just
that needed for stimulation
results in firing rate that is proportional to strength of
depolarizing stimulus over a broad range
-activated ( channel - allows for adaptation to signal
AMPA receptors - carries most of depolarizing current
responsible for excitatory PSPs (post synaptic potentials)
NMDA receptors - double-gated -glutamate must be bound
membrane must be strongly depolarized
o releases Mg2+
o Ml+ blocks resting chanhel, Ca2
+ nLong-term potential LTP) high activation Ca
2+ evels
activate protein kinases
Long-term depression LTD) modest activation Ca2+
ion concentrations
~ N a + low inside/high outside
- K+ low outside/high inside
- Ca2+ low inside/high outside
- Cl- low inside/high outside
Growth Guidanceneurotrophic factors released by targets guide nerve growth
cone
Eph to Ephrin, Ephrin to Eph, Semaphorin to Plexin
TR NSPORT
Highly Soluble
hydrophobic molecules 02 CO2 N2 steroids, hormones)
uncharged polar molecules H 20, urea, glycerol)
Low to no solubility
large uncharged polar moleculesions
Composition
- phospholipids asymmetrically distributed between layers
o helps w/ signaling, binding, living/dead discernment
- ffippase (p-type pump) - flip-flop diffusion
o moves phosphatidylserine
phosphatidylethanolanine to cytosolic leaflet
- l ipid rafts - high conc. of sphingolipids, cholesterol proteins
Pinocytosis - endocytosis of small parts of membrane forrecycling
Caveolae - caveolins - also capable of endocytosis
Transport
vesicles
Contain
Cargo proteins
Cargo protein receptors
Coat-recruitment GTPases
o Arf proteins - clathrin COPI assembly in Golgi
o SarI proteins COPli assembly in ER
Coat
COPI-coated - from Golgi
CGPII-coated from ER
clathrin-coated -Golgi <-> plasmal membrane <:->
endosomes
o coated pits - endocytosis
PIPs (phosphoinositrides) mark organelles membrane domain
dynamin - forms spiral around neck of bud - helps break.off buds
Rab proteins - guide vesicle targeting, on transport vesicles targ
membraneRab effectors
o membrane tethering - tether first attaches to Rab,
directing vesicle
o motor protein - propel vesicle along actin filament
SNAREs - recognizes proteins for targeting to membs/organelles
v-SNAREs - vesicle
t-SNAREs - target
NSF N - e t h y l m a l e i m i d e ~ s e n s j t i v e factor) separates SNAR
for reuse
secretory vesicles - concentrate cargo, activate by proteolytic
cleavage
synaptic vesicles - partially fused and primed for excretion, locally
recycled and reused
synaptotagmin - Ca2+ sensor, promotes rapid fusion of
docked synaptic vesicles w/ presynaptic membrane
ATP driven pumps
Hype - turb ine like, powered by electrochemical gradien
P-type phosphyrylate themselves during pumping
(Na+,K+,H+,Ca2+ gradients)
o Ca2+ pump Ca2+ ATPase) - sarcoplasmic reticulum
pumps Ca2+ into SR
o Na+-K+ pump (Na+ pump/Na+-K+ ATPase) -ATP driveantiport Na+ out, K+ in
V-type (vacuolar) - proton pumps that acidify membrane
bounded organelles (vacuoles, Iysosomes, endosomes)
ABC transporters-small molecules - ATP-binding cassett
o Mult idrug resistance MDR) protein - pumps
hydrophobic drugs out of cytosol
Overexpression gives cancer cells ability
pump out multip le drugs
Ion channels Na+,K+,Ca2+,Cr)
voltage-gated
o Na+ - skeletal muscle + nerve Na+ down its gradient,
create Action Potential
o K (delayed K channels) K out (down gradient)
returns nerve to negative potentia l
mechanically-gated
ligand-gated
o transmitter-gated - convert extracellular chemical
signals to electric
• excitatory neurotransmitters - open cation
channels, influx of Na+
• inhibitory neurotransrnitters - open CJ- or K+
• neurotransmitters are not exclusively inhibi tory
excitatory
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primarily excitatory -acetylcholine, glutamate,
serotonin
• inhibitory GABA (y-aminobutyr ic acid), glycine
o ion-gated, nucleotide-gated
& Transporters
tose transporter / permease proton-driven co-transporter
exchanger (antiport) - H+ out, Na+ in
+-driven Ci HC0 3- exchanger - Na+ & HC03- in, cr & H+ out
3- exchanger - HC03- out facilitates ql ick
of CO 2 in red blood cells ,
+-K- pump - Na + n, K out
leak channels - open even when unstimulated/resting
major-facilitator superfamily) - transport small solutes in
to ion gradients
bind ions for transport across membranes
- H20 pore, high cone. in epithelial cells of kidney
channel-coupled transmitter-gated ion channels ionotrop ic
- GTP-binding protein modulates interaction bin
& target
Most cell-surface receptors in animals are GPCRs
yme-coupled - function as enzymes or associate w/ enzymes
ar receptors - receptors w/ unknown ligands
cyclic AMP, az+, diacylglycerol
(GPCRs) largest family of receptors
Stimulatory G-protein (G,l activates adenylyl cyclase
Inhibi tory G-protein (G;) inhibits adenylyl cyclase +
regulates ion channels
t subunit - binds GDP & GTP
GRKs G-protein receptor kinases) - desensitize/inhibit
GPCR
Examples
rhodopsin receptor
6-adrenergic receptor
o ~ A R K - ~ - a d r e n e r g i c receptor kinase
• pps ~ - a d r e n e r g i c~ - a r r e s t i n ~ a r r ) binds to pp, triggers receptor
sequestrationacetylcholine receptor
. 0 muscarinic acetylcholine receptor (in heart muscle)
opening of K channels
Pathways
Gs adenylyl cyclase cAM PKA CREB + CREBB
DNA-binding
G inhibits adenylyl cyclase & ion channels
Gq 7 phospholipase C ~ { P L C ~ ~ IP3 inositoll;4,S
trisphosphate) + diacylglycerol (DAG) fcleaved from PIPzl
o IP3 7 IPrgated Ca2+ elease channels (ER) ~ C a z + +
calmodulin CaM kinase CREB + CREBB DNA
binding
o DAG arachidonic acid eicosanoids
prostaglandins (pain & inflammatory response)
Enzyme-coupled receptors
Most planet receptors are enzyme-coupled
receptor tyrosine kinases (RTKs) - growth factors
o most enzyme-coupled in animals are RTKs (rare in
plants)
o ephrins - cell surface bound extracellular signals
(bidirectional signaling)
o Eph receptors - cross-phosphorylation - creates dock
sites for signaling proteins (phospholipase C-y)
o Src & PI 3-kinase (phosphoinositide 3-kinase) - pps PH
(pleckstrin homology) where signaling complexes
assemble
o SH2 & SH3 - phosphotyrosine-binding (PTB) domains
tyrosine kinase-associated receptors depend on tyrosine
phosphorylation but lack kinase domain
o antigen & interleukins receptors in lymphocytes
receptor serine/threonine kinases
o most common receptor in plants - LRR (leUcine-rich
repeat receptor kinases)
o te trameric receptor complex of dimer'd type I & type
histidine kinase-associated receptorso two-component signaling pathway
o chemotaxis receptors - methylation responsible for
adaptation
receptor guanylyl cyclases
receptorlike tyrosine phosphatases
Desensitization
receptor sequestration - receptors removed from PM to
endosome(s)
receptor down-regulation - receptor destruction in
Iysosome(s)
receptor inactivation cytosolic domain is blocked
signaling protein inactivation intracellular signaling prote
is inactivated
production of inhibitory protein - pathway produces prot
that inhibits future signal
TR NDUCTION P THW YS
SMAD pathway
inhibits excess cell proliferat ion
T G F ~ (transforming growth factor
T G F ~ T G F ~ receptor II + G F ~ receptor I Smad3 + Smad4
DNA-bindingT G F ~ signal c a u s e s T G F ~ receptor II to pp T G F ~ receptor f
T G F ~ receptor I pp's Smad3
pp'd Smad3 binds Smad4 & is imported into nucleus
Inhibitors I-Smad, Ski, SnoN
Wnt signaling pathway
Wnt secreted signal proteins ( local mediator/morphoge
Frizzled cell-surface receptors
Disheveled scaffold protein
~ - c a t e n i n multifunctional protein
Wnt LRP Frizzled Disheveled -I destruction complex
catenin -I Groucho DNA-binding
lRP & Frizzled binding ofWnt activates Disheveled
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Disheveled inhibits destruction complex GSK-3)
~ 7 c a t e n i n inhibits Groucho, allowing LEF binding of DNA
signaling pathway
cytokine receptors - dimers or trimers
JAKs - Janus kinases
STATs - gene regulatory proteins
JAK -7 STAT-binding -7 pSTAT-7
-7 DNA-binding
cytokine brings JAKs together - pps each other, pp cytokine
receptor
STAT docks to pp'd cytokine receptor & pp'd by JAKspp'd STATs dimerize & enter nucleus
pathway
- RTK (receptor tyrosine kinase)
o Insulin receptor
o EGF receptor (epidermal growth factor)
- Ras - GTPase, acts as signaling hub
MAPK - mitogen-activated protein kinase
RTK -7 Grb2 -7 RasGEF SOS) -7 MAPKKK (Raf) -7 MAPKK {Mek -7
(MAPK (Erk) -7 gene regulatory proteins or protein kinases
signaling pathway
conserved developmental pathway
Delta signal- tells adjacent cells not to be neural
lateral inhibition neurons inhibit growth of
additional/redundant neurons
Delta -7 Notch receptor -7 TACE & Presenilin 1 -7 Notch segment
-7 D N ~ b i n d i n g
SIGN LING
of rhodopsin receptor
rhodopsino chromophore i i-cis retinal-light-absorbing
pigment
o opsin - protein portion
ll-cis-retinal is converted to all-trans-retinal
Ught -7 rhodopsin -7 Gt (transducin) 7 cGMP
p h o s p h o d i ~ e ~ i ' t S , , ~ , , ? - . J , . c G t \ I 1 P -7cGMP-gate ion channels close
-7AP
ylate cyclase converts GTP to cGMP
binds phosphorylated rhodopsin, inhibiting it
ins - 109
rod cells & 3 x 106
cone cells
& red cones distinguished environment of not
GPCR) - Golf - cAMP - cyclic-AMP-gated cation
Na+ influx
pain receptors that respond to heat, acidity,
of ions thought responsible for taste sensations
gustducin G protein associated w/ basic taste
Sound
ability to detect range of frequencies due to distinctions in basilar
membrane
PLASTIDS PHOTOSYNTHESIS
Proplastids - no internal membranes or chlorophyll
chloroplast photosynthetic organelle
chromoplast pigment organelleleucoplast storage plastid
o amyloplast stores starch (amylopectin) granule
Chloroplast
thought to have originated from cyanobacterium
thylakoid membrane -light absorption & electron transp
stroma chloroplast matrix
granum a)- stack(s) of thylakoids
make most of own lipids & some amino acids
mitochondria & chloroplasts are maternally inherited in
2/3rds of higher plants
o in some plants, inheritance is biparental
Neither ATP nor NADPH gen'd in chloroplast cancross
chloroplast membrane
Photosystems
LHCs - l ight harvesting complexes
exciton quantum of energy passed from excited molecu
to another
spatially distinct, located in thylakoid membrane (lamella
o PSlllocated on appressedside
o PSI located on nonappressed side
chlorophyll Mg2+ held in porphyrin ring
o P68 and P7 specialized chlorophyll molecules located
reaction centers2 photosystems required to evolve O2 (oxygenic
photosynthesis)
o plants, algae, & cyanobacteria - contain both PSI and PS
o purple bacteria and green sulfur bacteria w/ only 1
photosystem, cannot evolve O2
Photosystem 11- Type II (Pheophytin-QuinoneJ Reaction Center
most similar to the bacterial reaction center
higher energy to excite (shorter wavelength)
Structure
OEC (Oxygen-evolving/water splitting complex)
o 2 H20 -> 4H+ + 4e- + O
2
o passes electrons to P680
o Manganese center/complex - binds water
cytochrome b6-f complex - pumps H+ into thylakoid
space
pheophytin - chlorophyll lacking central Mg2+ ion
P680 -> pheophytin -> Po,. ->PQB (plastoquinones) -> cytochrome
b6f complex ->PQsH2-> cytochrome f -> r a t a i f u c ~ n -> PSI~ ~ ~ ~ ~Photosystem 1- Type I Fe-S) Reaction Center
does not contribute to proton gradient
generates NADPH NADP+ from stroma)
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-> A0 (Ao-) -> A1- (phyllquinone) -> 3x F e S ~ -> Fd (ferrodixin)+
+ H+
photophosphorylation
2 photosystems working in serial
-> plastoquinone -> cytochrome b6-f complex -> plastocyanin ->
-> ferredoxin -> ferredoxin NADP reductase
electrons cycle thru PSI reaction center
ATP formed w/ no net NADPH or O2
PSI -> cytochrome b6f complex '
accessory pigments that extend light absorption range
protect against damaging photochemical reactions reactive
oxygen species
8 carotene red-orange isoprenoid
lutein yellow blue light absorption
antennae of PSI of cyanobacteria red algae
proton pump only 247 amino acids long
sensory system
- allow plants to respond to light
phytochromes - cytoplasmic ser/thr kinases red ligh t
phototropin - blue light
cryptochromes - flavorproteins sensitive to blue light
CYCLE PHOTORESPIR TION
cyCle (carbon-fixation/assimulation)
- occursTn chforoplaststromaNADPH + 9 ATP + water -> glyceraldehyde 3-phosphate
2 -7-7-7 glyceraldehyde 3-phosphate)
Reactants Products
(3) CO2 + (3) ribulose 1,5 ~ (6) 3 ~ ~ o : ~ ~ ~ g l y c e r a t e1 bisphosphate
(6) 3-phosphoglycerate + ~ (6) 1,3-bisphosphoglycerate +
2 (6)ATP (6lATP
(6) 1,3- ~ Glyceraldehyde 3-phosphate +
3 bisphosphoglycerate (6) NADP+ + (6) i~ ? f>Jt\/ f
(5) glyceraldehyde 3- ~ (3) ribulose 5-phosphate + (2)4 phosphate i
(3) ribulose 5-phosphate + ~ (3) ribulose 1,5-bisphosphate
5 (3)ATP (first stable product)
(8 enzymes)
to plants)
to plants)
pentose phosphate pathway calvin cycle
Connects pentose phosphate pathway to glycolysis in anima
necessary for production of NADPH
fructose 6-phosphate + glyceraldehyde 3-phosphate ~ - 7erythrose 4-phosphate + xylulose 5-phosphate
sedoheptulose 7-phosphate + glyceraldehyde 3-phosphate
ribose 5-phosphate + xylulose 5-phosphate
Light Regulation of Calvin cycle·
disulfide bondsformthat immobilize catalytic activity
Enzymes
Reductiono ribulose 5-phosphate kinase
o glyceraldehyde 3-phosphate dehydrogenase - prevents
hexose synth competing w/ glycolysis in dark
Regenetation
o fructose 1,6 bisphosphatase
o sedoheptulose 1,7 bisphosphatase
RuBisCO (ribulose 1,S-bisphosphate carboxylase/oxygenase)
substrates - 0 2 C02 ribulose l,S-bisphosphate
requires 2 CO2 for normal function (1 t
as substrate, 2nd
for
binding site of metal ion cofactor)
Photorespiration
use of O2 instead of CO 2 in Calvin cycle
caused when CO2 is in low concentrations
wasteful use of energy w/o useful products
involves chloroplasts, peroxisomes, and mitochondria
rubisco's oxygenase activity
o ribulose l,5-bisphosphate 7 3-phosphoglycerate +
phosphoglycolate
carbons salvaged in glycolate pathway releasing
previously fixed CO 2
C4 plants (spatial separation)
isolates Calvin cycle to maximize CO 2 concentration(minimizing energy water loss of photorespiration)
mesophyl l cells
o CO 2 + phosphoenolpyruvate (PEP) -> oxaloaceta
-7 malate - to bundle-sheath cells)
• pyruvate phosphate dikinase - pyruvate 7 PEP
• phosphoerolpyruvate carboxylase CO
phosphoenolpyruvate (PEP) 7
oxaloacetate
bundle sheath cells
o malate 7 CO 2 + pyruvate
• CO2 -7 Calvin cycle
•pyruvate + Pi + ATP -7 AMP + PP
i+ PEP
(to mesophyll cells)
CAM (Crassulacean acid metabolism) plants (temporal separation
-night - CO 2 fixed into malate stored in vacuoles
-day stomata closed, malate source of CO2
dihydroxyacetonephosphate
formed in stroma transports ATP out of chlo-roplasts (ATP
cannot be exported on its own)
transported by the Pi-triose phosphate anti por ter
converted by glycolytic enzymes to 3-phosphoglycerate,
generating ATP in cytosol
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3-phosphoglycerate reenters the chloroplast, completing the
cycle
turned into starch in stroma
turned into sucrose production in cytosol
amylose al-4linked planar polymer of glucose
amylopectin - alA link highly branched polymer of glucose,
al-6 branch every 24-30 residues
synth'd in chloroplasts amyloplasts
o temporary storage chloroplasts of leaf cells
o long-term storage amyloplasts of colorless parts (seeds,
roots, tubers)
starch synthase - adds glucose to reducing end (donated by
ADP-glucose)
o Glucose I-phosphate + ATP j ADP-glucose
o ADP-glucose -(starch synthasej-j starch
ADP-glucose pyrophosphorylase
o regulates starch synthesis
o activated by 3-phospholycerate / inhibited by Pi
synth'd in cytosol of leaf cells
2 steps from U P ~ g l u c o s e + fructose I-phosphate
UDP-glucose -(cellulose synthase)-> cellulose
synthesis takes place in terminal complexes (rosettes)
rosettes simultaneously produce 36paraileiceHulosechains
DRI
catabolic processes (fatty acid oxid,) occur in the mito. matrix
inner membrane invaginations
- transfer across outer mem
- transfer acrOss inner mem - aided by memb potential
- helps jl-barrels fold in outer membrane
complex - moderates insertion of inner mem proteins that are
ndrial Hsp70 - acts as motor to pull precursor protein i nto
f?,;F I,,,
nsfer sequence prevents further translocation across inner
genome
Human mitochondria encode own ribosomal RNAs
-Plant mitochondria encode 5S mitochondrial rRNA, uses
standard genet ic code
membrane
most lipids imported modified rather than synthesized
high percent of lipids converted into cardolipin 4 fatty acid
tails)
impermeable to NADH (shut tle systems carry in reducing
equivalents)
proton gradient active transports:
o in pyruvate, Pi, ADP
o out ATP out
3 protein types
o electron transport chain
o ATP synthase
o metabolite transport proteins
Electron Transport (Respiratory) Chain
# Name Reaction e' carriers Notes
+
ADH dehydrogenaseNADH -7
FMN, FeSubiquinone
succinate-Q reductase / succinate -7FAD, FeS
not involved
dehydrogenase ubiquinone proton gradiubiquinone - mobile carrier (synth'd from benzoquinone [tyrosine
isoprene {acetyl-CoAl
Cytochrome b l
hemes,ill , complex ubiquinone -7 Rieske - 2 H
CoQ cytochrome c cytochrome cFeS, Rieske
instead of C
oxi do red ucta seISP
cytochrome c - mobile carrier - heme group, role in apoptosis
IV cytochrome coxidasecytochrome c -7 hemes;
O2 CUA CUB
ATP synthesis
Mitchel l chemiosmotic hypothesis - e' transport generates
proton-motive force that provides energy for ATP synthesis
Location - mito. Matrix, chloroplast stroma, cytosol in prok
ATP synthasome
o ATP synthase
• Fl - ATPase head + rotor stalk
• Fo - transmembrane H+ carrier / proton pore
o adenine nucleotide trans/ocase ATP/ADP antipor ter)
exchanges ATP/ADP ADP availability limits synthesis
o phosphate translocase symports H2P04- H+ into
matrix
o therrnogen (uncoupling protein) - energy of oxidatio
not conserved as ATP, but dissipated as heat
Shuttles
malate-aspartate shuttle (liver, kidney, heart) 2.5 ATP
NADH + oxaloacetate j NAD+ + malate j oxaloacetate +
NADH
reducing equivalents from cytosolic NADH transferred to
malate (malate transported replenishes NADH)
ions from malate, not malate transported across inner mito
memb:-r
g y c e r o I 3 ~ p h o s p h a t e shuttle (skeletal muscle + brain) 1,5 ATP
NADH to Complex III
converts reducing equivalents directly to ubiquinone in inn
mito. memb.
Oxygen Radicals
superoxide dismutase - antioxidant in cytosol, mitochondria
extracellular 2 02 + 2H+ 7 H20 2 +O2)
glutathione peroxidase reduce lipid hydroperoxides hydrogen
peroxide H 20 2 j H20)
CYTOSKELETON
ctin
Form
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examples: contractile ring (myosin II), muscle (myosin II),
stereocilia, microvilli
bindsATP/ADP
o Tform grow (ATP)
o D form - shrink (ADP) [cofilin - causes D-form actin
disassembly]
plus end - faster polymerization & depolymerization
actin capping - dramatically lowers critical concentration &
increases growth rate
Nucleated at cell cortext (just beneath PM)
nucleates most effectively when bound to existing actin
filament
branch from preexisting filament by ~ 7 0 °nucleate actin growth by capturing two actin monomers,
subfamily of Ras, regulate many aspects of actin & MT
ARP & formin)
bound to bound to GDls (guanine nucleotide dissociation
factors) when inactive
play part in cell proliferation, division, gene expression, &
apoptosis
Cdc42 - affects filopodia
Rac1 affects lamellipodia
RhoA - affects stress fibers
ERM family - mediates interactions between actin & PM
dystrophin - connects muscle fiber to EM thru PM
spectrin dimers
Actin arrays
contractile bundle - t1-actinincrosslinks
gel-like network - filamin crosslinks - important for cell
motility
tight parallel bundle - fimbrin crosslinks
proteins
myosin superfamily
o Structure:
• 2 heavy chains a-helixes coiled-coi l + 4 light chains
(51 fragment - myosin head)
• N-term - head/motor domain
• C-term tail
motor activity Regulated thru pp'tion
embrane protrusions
Activity
Types
actin filaments bend
arp2/3 nucleation of new filaments
actin depolymerization & polymerization
Filopedia - 1D projections
Lamellipodia - 2D - sheet-like extensions
Pseudopodia - 3D
Regulation
Rac superfamily (related to Ras) stims formation of
lamellipodia & stress fibers
o Activated by growth factors
Form
Consist of protofilaments made up of tubulin
a-tubulin & - ~ - t u b u l i n dimerize
o a oriented toward - ) , ~ oriented toward (+)
o u-boundGTP intra linksdimer (cannot be lost)
o ,-bound GTP inter-linksdimers (can be lost)
Microtubule Nucleation
critical concentration of tubulin, energy (37"C), & GTP
MTOC (microtubule organizing center)
y-TuRC y ~ t u b u l i n ring complex) nucleates microtubule
centrosome (single well defined MTOC in animals)centrioles - cylinders arranged in L shape at centrosome
center
centrosome matrix/pericentriolar material
spindle pole body (MTOC in fungi & diatoms)
imbedded in nuclear membrane
microtubules nucleate ll aroundnuc/eus in plants
Microtubule motor proteins
Kinesin mostly toward +) 2 heavy chains, 2 light chains
one family C-term head, walks toward (-)
o Hand over hand motion - never completely dissociate
o Nonclaret disjunctional(NCD) toward (-) kinesin
Dyneins - toward (-) - 2-3 heavy chains + variable
intermediate & light chains
o Largest & fastest molecular motors
dynactin protein complex
links cargo (organelles, chromosomes) to dynein & kinesin
Associated proteins
M Ps (microtubule-associated proteins) bind alongside MTs
mediate interactions b/w MTs & other components
Tau & MAP2
plectin crosslinks actin, MTs & intermediate filaments
catastrophe factor increases chance of microtubule catastrophe
Mitotic Spindle
Centrosome - spindle pole
MTs
o astral interact w/ cell cortex - dynamic instability
o kinetochore - attach each chrom to each spindle pole
o interpolar - hold halves of spindle together by overlapping
Kinetochores attach MTs ~ 1 5 ) to chromosomes
in animals, position of contractile ring determined by signals from
anaphase spindle
poleward flux - net addition of tublin to +), loss at Haverage lifetime of microtubules during mitosis is 60 90 seconds
Ran-GTP & GEF bound to chromatin, stabilize MT spindles
Cilia flagella
Axoneme 9 doublet MTs (A-full, B-partial) arranged arou
2 MTs
Dynein responsible for MT sliding during flagellar bendin
Flagella = bacterial flagella (made up of flagellin, not MTs)
Basal bodies kinetosome) firm root of most cilia & flagel
Neurons - single MTs do no extend over the length of
axons/dendeites overlap
Dendrites - MTs of mixed polarity
Intermediate filaments
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_ tetrameric,disassembly triggered by subunit phosphorylation
keratins - epithelial cells (hair, horns, etc)
o high % of glycine, alanine, & cysteine (14%)
o heterodimers of type I (acidic) & type II (neutral/basic)
o important in cell adhesion (in desmosomes)
desmin - heart muscle
neurofilaments - highly concentrated in axons
Y LE
yokinesis - nuclear div w/o cytoplasmic
of H1 & Rb limit replication to once per cell cycle
point - replication commitment (during GI )
checkpoint - triggers events leading to chromosome
sister-chromatid separation (unattached kinetochores inhibit
separation)
- specialized nondividing state
terminally differentiated Go - cell-cycle control system
completely dismantled
in G1
assembly of pre-replication complexes at origins
degradation of S-phase cyclin-Cdk inhibitor
activation of pre-replication complexes
APC/C activity
o degradation of securin
o degradation of mitotic cyclins
G1Cdc1 binds to APC/C - prevo inhibited by pp by M-cyclin-Cdk
Cdc1-APC/C activator
CKI accumulation + decr. cyclin expression suspend Cdk activity
S-cyclins - trigger progression through star t/rest riction point
- hours in mammals
synth increases during G2 & M due to increased transcription
Activated by Cdc25 removing inhibitory pps (by Wee1)
- activated by dephosphorylation at onset of mitosis
activates Cdc25 & inhibits Wee1
Induces assembly of mitotic spindle
Insures at tachment of sister chromatids to both poles of
spindle
Triggers chromosome condensation
Promotes breakdown of nuclear envelope (in animals)
Aided by pole-like kinases & aurora kinases
APC/C)
ubiquitin ligase
Activating subunits
Cdcl activation during late mitosis & early G1
o
o
inactivates Cdks & destroys geminin (allowing fo
pre-RC assembly)
pp d by M-Cdk allowing Cdc20 to bind
Cdc20 - activating subunit of cyclosome dur ing anaphase
(increased transcription as cell a'pproaches mitosis)
o Targets securin, S & M-cyclins for destruction (lo
of most Cdk activity)
dk Regulation
phosphyrlation of Cdk-subunit
o Cdk-act ivating kinases CAK) - fully activates cyc
Cdk complexo Cdc25 - removes i nhibitory pp'tion, increaSing C
activity
Binding of CKls (Cdk-inhibitors)
o Wee1 inhibits cyclin-Cdk complex
Proteolysis of cyclins
Changes in gene expression of Cdk regulators
Securin - binds to and inhibits separase - protease which cleaves
cohesion
Control of Cell division & Growth
Mitogens - stim Cl/S-Cdk activity & cell divisiono Ras GTPase) - activates MAP kinase cascade - Myc &
other gene reg. proteins
o G1 > G1-Cdk -> E2F proteins - G1/S-cyclins / S-cyclins
DNA synthesis
Growth factors - stim cell growth (increase in mass) -
promotion of synthesis & inhibition of degradation
o TOR - stims S6 kinase, elF4E (initiator factor)
o Promotes gene expression of ribosomal subunits
Survival factors - suppress apoptosis
DNA damage-7 ATM/ATR kinase -7 Chk1/Chk2 kinase -7 p53 -7 p2
binds G1/S-Cdk & S-Cdk
Mdm2 - degrades unphosphorylated p53
Stages o M Phase
(1) prophase - chroms condense, spindle assembles bin
centrosomes
(2) prometaphase - breakdown of nuclear envelope, chroms atta
to spindle
(3) metaphase - chromosomes align at equator, sister chromatid
attached to opposite poles
(4) anaphase - chromosome segregation - synchronized separat
of sister chromatids
anaphase A - shortening of kinetochore MTs & movement o
chromosomes along MTs towards polesanaphase B - sliding & elongation between interpolar MTs,
pulling force of astral MTs pull centrosomes apart
(5) telophase - chroms arrive at poles & decondense, nuclear
envelopes develop
o depp of proteins responsible for reassembly of nucleus
(6) cytokinesis - contractile ring (actin + myosin) divides cytoplas
Prophase I
leptotene - homologs condense & pair, recombination begins
zygotene - synaptonemal complex develops at recombination site
pachytene - assembly complete, homologs synapsed along enti re
length (can persist for days or longer)
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disassembly of synaptonemal complexes & chromosome
- transition to metaphase I, thromsdetach from nucleus
- period lw meiotic divisions
b nd - marks site of cell wall assembly in plants
- precursor to cell wall formed on interpolar MTs in
OPTOSIS
- inactive precursors proteolytica lly cleaved by other
cleavesd in two spots large & small subunit - dimerize & form
tetramers
Prodomains I caspase recruitment domain - discarded
~ y s t e i n e active site & cleave at aspartate c-asp-ases)
Init iator procaspases - initiat ion executioner procaspases
Executioner procaspases cleave other EPs & target proteins,
cleaves protein that inhibits endonuclease
lAPs inhibitors of apoptosis) - inhibit caspases
Anti-lAP - bind to lAP preventing their interference wi
caspases
p thw y
extracellular signaling proteins bind to death receptors
Fas receptor binds Fas ligand
procaspases bind to death domains of Fas forming DISC
(death inducing signal complex)
FLIP - competes w/procaspase for binding on death domains,
lacks proteolytic activity
release of mitochondr ial proteins into cytosol some activateproto ytic caspase cascade
Cytochrome c - binds to adaptor protein Apafl (apoptotic
protease activating factor-i
Apoptosome heptamer of cytochrome c +Apafl
proteins regulate release of cytochrome c
BHl-4 anti-apoptotic, binds 1-4 (Bcl2 homology 1-4)
Bcl2 & Bcl-XL on cuytosolic surface of mit.mem.
BH123 pro-apoptotic, binds 1-3 - aggregate to pump out
cytochrome c
Bak - bound to cytosolic surface of mito.mem.
Bax - cytosol, transferred to Mito.mem. when active
BH3-only pro-apoptotic, inhibit anti-apoptotic
proteins
P53 - stims expression of BH3-only genes
stim expression of Bcl2
Uninhibit Bcl2 from BH3-only Bad, etc)
Uninhibit lAPs (pp'ing anti-lAP (Hid, etc)
- cleaves sphingomyelin into ceramide,
to be part of apoptosis
- condensed appearance of DNA indicating apoptosis
Cell swells wi water & ruptures
turmor necrosis factor 7 diacylglycerol 7 sphingomyelin
breakdown
CELL DHESION
Cell Cell Anchoring Junctions
Junction name Internal External
adherens cadherin actin
desmosomes cadherin intermediate filament
Cadherins - in animals, not plants or bacteria
- Depender:lt on ci+- Generally like-like binding
adhesion belt - cad herins anchor proteins + actin
Desmosomes contain the transmembrane cadherin proteins
desmoglein and desmocollin
Occluding Junctions
I Junction n me Organisms Proteins
I Tight junctiorl vertebrates claud in & occludin
I Septate junct ion invertebrates
fectins proteins that can bind carbohydrates, read sugar code
- sefectins - mediate cell-cell recognition
Immunoglobulin superfamily proteins mediate ci+ independent
cell-cell adhesion
- ICAMs intracellular cell-adhesion molecules
VCAMs - vascular
NCAMs neural
Junctiional complex - series of unctions between epithelial cells
tight, adherens, desmosomal
Signal-relaying junctions
Gap junctions - connect cells electrically & metabolically
o Connexins
o Coordinate activities of adjacent cells through sharing of
metabolites & ions
• Synchronize muscle contractions
• Important part of embryogenesis
o Permeability regulated - dopamine & Ca2+ (reduce or clos
Plasmodesmata - desmotubule - continuous wi ER
Chemical & Immunological synapses
o Scaffold proteins - important for cell-cell adhesion at
synapses
Cell polarity - dependent on membrane-associated proteins: Par3
Par 6 (scaffold proteins) & atypical protein kinase C aPKC)
Cell Matrix Anchoring Junctions
Junction name Internal External
actin-linked matrix integrin actin
adhesions
Hemidesmosomes integrin intermediate fila m
Integrins
- mUltiple forms from multiple genes
- a + 3 subunit clamps onto matrix
talin - binds integrin to actin
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ronectin - glycoprotein that aids in matrix.-cell interactions
through Integrins
dependence - cell proliferation & survival tied to binding
adhesion kinase FAK) - cross-pp - docking site for
tyrosine kinases
Number of focal adhesions regulate level of adhesion/motility
Matrix
Synth & maintains EM
can differentiate into
o bone cell (osteoblast/osteocyte)
o fat cell (adipocyte)
o cartilage cell (chondrocyte)
o smooth muscle cell
EM
rich in nitrogen-containing polymers
excreted largely by fibroblasts
o molecular filter - proteoglycan key
o cell segregation
o scaffold
o regeneration - tough, often remains inact, guides repair &
regeneration
o fibrous proteins (usually glycoproteins)
o laminin - 3 chains, shaped like cross
o Type IV collagen - ropelikesuperhelix
o nidogen
glycosaminoglycans GAGs) + core proteins
EM
(Glycosaminoglycan) - unbranched polysaccharides composed
repeating disaccharide units 1 an amino sugar)
o Hyaluronan - simplest GAG, spun out directly from cell
surface by embedded enzyme complex
major proteins of EM
exported from ER as folded triple helix
repeating tripeptides (glycine-x-y) rich in glycine, proline &
hydroxyproline
mature collagen does not contain cysteine or disulfide bonds
o fibrilla r collagens - Type I principle coHagen of skin & bone
o Fibril-associated collagens -IX & XII- retain propeptides
organize fibrils
o Network -forming collagen - IV
o Anchoring fibrils - VII
tic fibers elastin - hydrophobic
Covered by sheath of microfibrils that act as scaffolding
fibrillin
lfate - present in all animal cells
common sugars:
o N-acetyl-O-glucosamine
o O-glucuronic acids (O-xylulose 5-phosphate 7 pentose
phosphate pathway
o L-iduronic acid
Matrix degradation
Matrix metalloproteases - depend on bound Ca2+or Zn
2+
Serine proteases - highly reactive serine active site
Plant Cell walls
almost no nitrogen
microtubular - cortical array determines orientation o
cellulose synthase
Primary cell walls
thin, extensible walls of newborn plant cells
o cellulose microfibrils interwoven w/ network of pectic
polysaccharides
o Pectins - negatively charged branched polysaccharide
Secondary cell walls
o rigid wall formed by deposit ing new layers of matrix inside
old ones
o Lignin network of phenolic compounds
Calmodulin (CaM) - calcium-binding protein - binds 4 Ca2+
responsible for regulatory ability of ci+F h nd - helix-turn-helix found in Ca-binding proteins
major classes
fibronectin Ig-superfamily CAMs, P-selectins
CaM kinases I-IV Ca2+/calmodulin dependent protein kinases)
serine/threonine kinase
phosphorylates self (memory)
DEVELOPMENT DIFFERENTIATION
Hoxcomplex
important switches that control cell identities by controlling
transcription target genes
carries permanent record of positional information
controls anteroposterior axis
Mammals have 4 Hox complexes
Morphogens
long-range inducers that exert graded effects
Hormonal signals that shape development, imposing a
pattern a whole field of cells
Induces graded responses, new gene expression, differentiat ionevents, & change in cell fate
Embryological Deve lopment
Concepts
Synctium - cell w/ multiple nuclei
Cel/u/arization subdivision of synctium so that each cell has only
one nucleus
Structures
embryonic disk ectoderm + endoderm
Blastula - hollow ball of cells, with an internal fluid-filled cavity
inner cell mass gives rise to extra-embryonic structures
hypoblast & trophoblast
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- invagination that circle vegetal pole
Organizer (Spemann'sOrganizer) dorsal lip of blastopore
which organizes formation of main body axis
o n o t o h o r d ~ o u t g r o w t h of organizer-that defines central
body axis
ulation - transformation of simple ball or hollow sphere of cells
structure with a gut
3 germ layers formed
ectoderm - exterior, precursor of epidermis and nervous
system
o neural tube
endoderm interior, precursor of gut and its appendages' (lungliver)
mesoderm cells blw ectoderm endoderm; precursor of
muscles, connective tissues, etc.
o notochord defines primitive axis of embryo
o somites segments (cohesive groups of celis, separated
by clefts)
• form along neural tube dermis, skeleton, muscle
(heart)
serially repeating segments
groups of cells that are set aside, apparentlytiated, in each segment of the larva
n clock repetitive alternating pattern of gene
strategy for generating a progressively more
of few basic themes
genes
gap - divides embryo into broad regions
pair-rule segment polarity defines segment stripes
homeot ic - specifies which organs appendages develop
polarity - established as early as oogenesis in some species
Left-right polarity
o controlled by odal in embryo
Anterior-posterior patterning
o in limb bud Shh gradient
o in developing spinal chord - BMP, netrin hh
id - anterior
os - posterior
Carcinomas - epithelial cells
Sarcomas - connective tissue
Leukemias - blood or bone marrow
Lymphomas lymphocytes
of cancer growth
more self-sufficient
o high glucose uptake metabolism
o insensitive to anti-proliferative signals
o less prone to apoptosis
o induce help from stromal cells
o induce angiogenesis - format ion of new blood vessels
o produce telomerase or other method to stabilize
chromosome length
o reduced growth factor requirement
altered morphology
o metastasize
o genetically unstable
o loss of actin microfilaments
o defective differentiation
o defective cell cycle braking
protein kinases are found to be overactive in cancer cells
Types of carcinogenstumor initiators - damage DNA
tumor promoters - do not damage DNA
Cancer-critcal genes
proto-oncogenes - cancer risk from over activity - fos, myc
ras often g rowth factors and growth factor receptors
tumor suppressor genes - cancer risk from too little activit
DNA maintenance genes - mutat ion results in genetic
instability
Src - proto-oncogenic tyrosine kinases
Ras - g protein; mutated in 1 in 5 cancersRb - tumor suppressor
Myc - transcription factor I activator
Cancer Diagnosis
Intermediate filaments - may be used to diagnose tissue of origin
mes test assay to assess mutagenic potential
IMMUNOLO Y
pathogens
obligate pathogens can only replicate inside the body of their ho
and are calledfacultative pathogens replicate in wate r or soil, cause disease if
they encounter susceptible host
opportunistic pathogens normally harmless, cause disease in
injured or immunocompromised host
vaccination enhanced function of B cells cytotoxic T cells
autoimmune disease subverts immune system by destroying
helper T cells
transformation uptake of naked DNA
transfection introduction of foreign DNA by non-viral method
transduction foreign DNA introduced via phage vector
conjugation genetic material transferred unidirectionally from
bacterial donor to recipient
recipient F
donors
o F piliated w conjugative plasmid
o F - conjugative plasmid contains chromosomal genes
o Hfr - conjugative plasmid incorporated into chromosome
rolling circle (sigma) replication
Bacteria
classified by shape (rods, spheres, spirals) Gram-staining
properties
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o activate endothel ial cells - cause monocytes & neutrophils
in the blood to adhere
o chemokines - positively charged cytokine that directs
migration of monocytes & neutrophils to infection
. TH2 cells
secrete cytokines interleukins 4 and 10 lL4 and ILlO)
o stim B cells to make antibodies (lgM, IgA, IgE, & some IgG)
cytotoxic T Cells
induces apoptosis in infected cells
o Fas ligand - binds receptor initiating caspase
cascade
per/orin - pore-forming protein r e l ~ a s e d by cytotoxic T censcross-presentation - activation by uninfected dendritic cells
regulatory T cells - suppress activity of other T celis
large role in humoral immune system
make receptor & secretory antibodies (expanded ERs)
plasma cells
memory B cells - form following primary infection
megakaryocytes
o responsible for platelets
o extraordinari ly large w highly polyploid nucleus,
remains in bone marrow
Receptors
TCRs) - antibody-like heterodimers
immunological synapse - interface lw antigen-presenting
cell & lymphocyte
diversity thru D-to-D joining, junctional imprecision, & N-
nucleotide addition
receptor TLR) - pattern recognition receptors that triggers
immune response to pathogens (microbial DNA, etc.)
proteins - exclusively intracellular pattern recognition
major histocompatibility complex)class I MHC - on all cells, display peptides from cytosolic
proteins
o a chain & ~ 2 m i c r o g l o b u l i n CD8)
o usually bind to cytotoxic Tcelis
class II MHC - macrophages, dendrit ic & B celis, display
endocytosed peptides
o usually bind to helper & regulatory T cells CD4)
Ig) - 2 heavy & 2 K & A light chains,
tail & Fab binding site
Primary classes
IgM - Il heavy chain - monomer or pentamer; first & major
antibody in primary immune response, activates complement
IgD - < heavy chain - monomer; second class to appear on
surface of B celis
Secondary classes
IgA - a heavy chain - monomer, dimer or trimer; principle class
in secretions (saliva, tears, milk)
IgE - E heavy chain - monomer; allergic response, binds mast
cells in tissues & basophils in blood (allergic reactions)
IgG - Y heavy chain - monomer; major antibody in secondary
immune response, most abundant in blood; activates
complement
o tail binds to Fc receptors on tnacrophages & neutrophils
class switching - after stimulation, B cells switch from IgM & IgD to
other antibodies (does not involve l ight chains)
affinity maturation - antibody affinity for antigen increases over ti
polyclonal antibodies - produced by many different B cells
responding to one antigen that bind to different epitopes
monoclonal antibodies - identical antibody produced by identical
cell in response to one antigen
V D)J recombination
light-chain V region V + J 7 VJ)
-long V gene segment
- short J gene segment joining)
heavy chain V region 0 + J 7 OJ + V 7 VDJ)
0 gene segment diversity)
. - short J gene segment joining)
-long V gene segment
RSS recombina tion signal sequences) - before Js & after Vs
RAG recombination activating genes)
-cuts in gene segments & recombination signal sequences
- recruits rejoining enzymes (DNA double-strand repair)
junctional diversification - random loss and gain of nucleotides at
joining sites of antibody gene segments
allelic exclusion - choice between maternal & paternal chromosom
loci
activa tion-induced deaminase AID) - deaminates C to U in V regio
producing mismatches, repair produces mutations
somatic hypermutation - accelerated mutation rate in B celis
Self-tolerance
central tolerance
receptor editing self-reactive lymphocytes change their
antigen receptors
clonal deletion, self-reactive lymphocytes apoptose
peripheral tolerance
clonal inactivation (clonal anergy), self-reactive lymphocytesfunctionally inactivate
clonal suppression regulatory T cells suppress sel f-reactive
lymphocytes
METHO S
DNA librar ies
genomic DNA l ibrary- collection of plasmids containing genome o
organism (not necessarily in whole genes)
cDNA l ibrary- collection of plasmids containing cDNA only (DNA
complementary to mRNA), not full genome
o contains info on level of varying expression - some cDN
present in duplic it
o contains unin terrupted coding sequences
Mutation methods
Site-directed mutagenesis - DNA is cut, DI\IA is inserted
Oligonucleotide-directed mutagenesis - complementary segment
made w single base-pair mismatch
hybridization & replication result in site mutation
induced somatic mutations - modification of DNA in subset of celi
at late stage of development
genetic mosaic- organism subjected to induced somatic
recombination
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fingerprinting - does not normally use DNA microarrays
number of tandem copies of a simple sequence repeat
uses PCR, restriction endonuclease digestion & southern
blotting
RFLP - restriction fragment length polymorphism
microdissection
use of laser to move organelles, chromosomes
- images from a series of focal planes are combined to
an in focus plane
(up to .2 11m)
confocal- excludes out of focus light
o multiphoton confocal- uses multiple photon absorption
for greater penetration
fluorescence microscope - uses fluorescent dyes
,. TlRF total internal reflection fluorescence) - use of indirect
light to image cell- visualization of single molecules
Transmission (TEM) (up to Inm (10 angstroms) for biological
specimens) [0.1 nm for non-biological]
o difficult preparation/fixing - fixed, dehydrated, resin-
embedded
o supercooled into vitreous ice (noncrystalline glass )
Scanning SEM) (up to 10nm res)
o 3D structure of surface (10nm res) - used for cells & tissues
not subcellular components
Techniques
immunogold - gold particles attached to antibodies
metal shadowing - heavy metal evaporated onto specimen
from oblique angle
o negative staining - additional step to invert image
crystallography - EM + electron diffraction analysis
cryoelectron - specimen can be viewed w/o fixation, staining,
drying (cryogenic container at -160C)
o cryostat - cuts thin slices of specimen
single-particle reconstruction - thousands of images of
identical molecules are arranged together
tomography- 3D through rotation of specimen in TEM and
combination of images
atomic force - allows manipulation of individual molecules w/
silcon probe
spectrometryprecise determination of the mass of a protein (mass used to
ID unknown protein)
determine length of hydrocarbon chains or position of double
bonds
Techniques
Matrix-assisted laser desorption ionization MALOI) - soft
ionization process used on biomolecules
Time of Flight TOFMS) - mass-charge ratio of ions used to ID
molecules
TOF/TOF - tandem mass spectrometer - change in mass of IDs
amino acid lost (leucine/isoleucine same mass)
Type I - cleave DNA at random sites (up to 1000 bp from
recognition site)
Type cleave DNA within known recognition sequence
Type cleave ~ 5 b p from recognition site (requires ATP)
isoschizomers - restriction enzyumes from different species w
same recognition sequence
peR (polymerase chain reaction)
single-stranded DNA primers
deoxynucleotide triphosphates (dNTPs)
dideoxynucleotide triphosphates (ddNTPs)
stringency-level of homology; fidelity & specificity of hybridannealing
fidelity - depends on temp, pH, [NaCI], & probe sequence
U:;R ligase chain reaction) - good for detection of alleles, point
mutations
Vectors
plasmids 5,000-400,000 bp - dif ficult to clone segments long
than 15,000 bp, mUltiple per cell
o cosmid - have euk & prok initiation sequences
phages 40,000-53,000 bp - ID by plaque formation
BACs (Bacterial Artificial Chromosomes) - plasm ds for clonin
DNA of 100,000-300,000 bpo Accommodates largest inserts (up to 300k bp)
o from F plasmid, 300,000 - Imil bp, 1-2 copes per
YACs (Yeast Artificia l Chromosomes) -l inear, telomere stabl
longer than 150,000 bp
o contain features necessary for propagation as an
independent chromosome (centromere, telmomeres, o
o Plasm ids for yeast transformation - up to 2,000,000 bp
o Not recircularized, remain as linear molecules
Shuttle vectors - plasm ds that can be used to transform
different species
Expression vectors - cloning vector w/ transcription &
translation signals needed for regulated expression
o plasmids designed to produce large amounts of mRNAo contains promoter , ribosome-binding site, & transcriptio
termination sequence
Viral vectors - used in animals for transformation
Delivery of non-permeable substances - vectors, etc)
micropipette injection - injection of DNA directly into cell
and/or nucleus
electropora tion - electric shock leaves bacterial cell membra
temporarily permeable to DNA uptake
membrane-enclosed vesicles
gold particles - shot into cells (DNA coated)
Fluorescence
GFP (Green Fluorescence Protein)
FRET (fluorescence resonance energy transfer)
cellular location of reactions
FACS (Fluorescent-activated cell sorting) - used to separate cells b
DNA content, size, surface markers
FRAP (fluorescence recovery after photobleaching) -laser beam
extinguishes fluorescence --recovery recorded
Electrophoresis
SDS (sodium dodecylsulfate) - determines purity and
molecular weights of proteins
o SDS treated proteins - smaller weight travels faster
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o anomalous if protein contains carbohydrates in large %
isoelectric focusing - organic acids and bases establish pH
gradient across gel, target protein migrates to pH pi
two-dimensional use o fboth SDS& isoelectric focusing
pu Ised field gel electrophoresis - alternating voltage allows for
better resolution of large DNA molecules (>15-20kbs)
gel-mobility shift assay - detects DNA bound proteins through
retardation of movement
denaturing polyarylamide - used in DNA sequencing
electrophoresis; probing & hybridizaton in RNA/DNA,
y affinity in proteins \
allows detection of minor sample component and provides
estimated weight
target sequence usually twice as long as probe
o southern - DNA
o northern - RNA
o western (immunoblotting) - protein
reservoir - container
stationary phase column
mobile phase - solution to be run through column
effluent - solution passed thru columnTypes
cation-exchange - negatively charged stationary phase
hydrophobic hydrophobicity
gel fil tration / size-exclusion - separates according to size
(smaller proteins slowed down by pores/cavities)
affinity - functional groups in stationary phase bind to proteins
w/ varying affinity
HPLC (high performance liquid) - high pressure
chromatography that limits diffusional spreading maximizing
resolution
absorption or thin-layer - separates lipids of diff polarity
techniques
effective for detecting & quantifying hormones (can detect
very small amounts of specific material)
Techniques
RIA Radio Immuno Assay) quantifies amount of hormone in
sample
o incubate sample w/ hormone-specific antibody & purified
radiolabeled hormone
o unlabeled hormone competes & displaces radiolabeled
hormone from antibody
ELISA enzyme-linked immunosorbent assay) - screens for
presence of an antigen
Protein micro array / chip array of antibodies to ID proteinsCoimmunoprecipitation precipitation of complexed proteins
w/ targeted ones
epitope tag - use of known epitope to tag protein
fused cells w/ separate nuclei
- fused B lymphocyte w/ transformed tumor line cell) B
hocyte - molecular factory for monoclonal antibodies
/ dideoxy method - ddNTPs terminate DNA synth, creating
of various lengths sequenced thru electrophoresis
based on chemical modifications of DNA
Edman degradation label & removal of 1 AA at time (res: AA
sequenator automated Edman degradation
chromosome walking - sequencing thru cyclic creation of primers
Protein Sequencing
TOF/TOF - tandem mass spectrometer - change in mass of IDs
amino acid lost (leucine/ isoleucine same mass)
Sequence evolution (species relatedness)
Blosum62 (62% identical) - blocks substitution matrix, mathemati
analysis of genome evolution
Protein interactions
.Surface plasma resonance SPR) detects binding interactions by
ch,ange in resonance angle, good for timing of interactions
yeast two-hybrid system
IDs protein interactions w/ known bait ), co-activators & c
repressors
reporter genes easily assayed enzymes
Protein conformation
NMR spectroscopy - protein conformation
Genetic Analysisgenetic screen - genetic analysis of numerous individuals to ID
mutant allele responsible for mutant phenotype
pleiotropy - single gene influences multiple phenotypic traits
epistasis analysis - analysis of combinations of mutations in orde
determine order of gene action (epistatic - occurring prior)
complementation test - tests if 2 mutations are products of
mutations in one or two genes
Cre-Lox Recombination - gene knockout: lox sites - target gene, cr
recombinase remove it
protein synthesis - AAs - on polymer support
Gene Expression
DNA microarrays - rapid + simultaneous screening of thousands o
genes wIn species
probed w/ mRNA or cDNA ID what genes are being expres
Comparative genomic hybridization CGH) - DNA microarray
comparison to see what genes over/under expressed
DISORDERS TOXINS REAGENTS
DRUGS
Disorders
Huntington's disease - autosomal dominant disorder
Parkinson's disease - underproduct ion of dopamine
maple syrup urine disease lack of debranching AA deg. Enzyme
abnormal brain development & infancy death
anemia - hemoglobin deficiency
beriberi -lack of Vitamin B1 (thiamine) - involved in breakdown o
glucose
hypoxin -lowered oxygenation of peripheral tissues
Jaundice accumulation of bilirubin in blood b/c not enough
glucuronyl bilirubin transferase to process to bilirubin diglucuron
diabetes - failure of insulin secretion or action, fail to synth fatty
acids from carbohydrates or amino acids
acidosis & ketosis lowered blood pH caused by ketone bodies
(acetoacetate D-j3-hydroxybutyrate) in blood
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sease - autosomal dominant disorder
kinson's disease - underproduction of dopamine
emia - hemoglobin deficiency
- lack of Vitamin B1 (thiamine) - involved in breakdown of glucose
-lowered oxygenation of peripheral tissues
ndice - accumulation of bilirubin in blood blc not enough glucuronyl bilirubin transferase to process to bilirubin diglucuronide
etes - failure of insulin secretion or action, fail to synth fatty acids from carbohydrates or amino acids
-lowered blood pH caused by ketone bodies (acetoacetate D - ~ - h y d r o x y b u t y r a t e in blood
(death caps) inhibits RNA polymerase
inhibit MT assembly by capping ends(blocks cell division), induces polyploidy in plants
CO, azide blocks electron transport from Complex IV (cytochrome oxidase) to O2
blocks elongation
DNP) dissipates proton gradient in mitochondr ia, eliminating ATP synthesis
inhibits CAC
inactivates EF-2, blocking elongation
(death caps) prevent actin disassembly
TAG) in skeletal muscle & liver can be toxic
purine analog that blocks purine breakdown, lowering uric acid levels
aspirin (NSAlDs) - inhib its cyclooxygenase COX), essential for arachidonate conversion to prostaglandins & thromboxanes
prednisone - inhibits arachidonic acid release by phospholipase A2 blocking prostaglandin formation
inhibit Na+,K+-ATPase (l [Na+], l [Ca2+]), increase contractile force
prevents elongation, inhibiting transcription
inhibits oxidation of CoQ by binding Complex III (cytochrome bCl complex)
binds 50S subunit, preventing translocation
blocks Fa (proton pore) of ATP synthase
causes premature release of protein chains, inhibiting synthesis
binds 30s subunit, preventing initiation
prevents synth of peptidoglycan cross-links in cell wall of gram-positive bacteria
prevents release of EF-G-GTP, blocking tranlocationinhibits peptidal transferase activity of bacterial ribosomes
binds 16S rRNA, blocking aminoacyl-tRNA
& performic acid oxidizes disulf ide bonds
chloride used in n-terminal AA analysis
used to cleave peptide chains at methionyl residues
used to modify cysteine side chains into thioethers, preventing disulfide bond reformation
& chloroform precipitate proteins out of lysate
Intercalating gents
o Planar molecules that lodge blw stacked DNA bases causing DNA pol to insert or skip bases
o proflavin acridine organe & ethidium bromide
base analogs
o less stable, causes mismatches
o 5-bromouracil & 2-aminopurine
alkylating agents cause mismatch by chemically modifying bases
o hydroxylamine GC 7 AT
spermatocyte -(mei-I)--7 sec. spermatocyte -(mei-II)--7 spermatid - spermatozoa (mature sperm)
-(rep.)--7 prim. oocyte -(meios is 1)--7 sec. oocyte -(mei-II)--7 oovid - ovum (mature egg)
PLP) - Schiff base + E-amino
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