医学史简论( 5 ) A Brief History of Medicine Yu Hai Zhejiang University School of Medicine.
Molecular Biology in Medicine 医学分子生物学
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Transcript of Molecular Biology in Medicine 医学分子生物学
The greatest intellectual revolution of the
last 40 years may have taken place in biology.
Can anyone be considered educated today
who does not understand a little about
molecular biology?
─F. H. Westheimer (Harvard University)
Genetic Information Transfer
遗传信息的传递 Gene Transcription 基因转录 RNA Splicing & Editing RNA 剪切与加工 Protein Synthesis & Processing
蛋白质合成与加工 Regulation of Gene Expression
基因表达的调控 ( 包括 miRNA 、 RNAi)
分子生物学主要内容
分子生物学主要研究技术 分离、纯化(主要是生物大分子) 克隆、表达 PCR (多聚酶链式反应 ) 凝胶电泳:琼脂糖凝胶电泳; SDS -聚丙烯酰胺凝胶电泳 ( SDS-PAGE );等电聚焦电泳;双向电泳 印迹技术: Southern blotting; Northern blotting;
Western blotting
微阵列技术: genechip, microarray, protein chip
基因操纵技术: Gene knock-out/knock-in
RNA interference (RNAi)
分子生物学主要研究技术 蛋白质相互作用:酵母双杂交、免疫共沉淀( Co-IP )、 pull-down 、 FRET 、表面等离子共振技术 (SPR)
蛋白质鉴定:质谱 蛋白质与核酸相互作用: ChIP 、 ChIP-on-chip
研究生物大分子三维结构常用的实验手段: X 射线晶体学、核磁共振、电子显微学、原子力显微镜 以及 X 射线小角散射等。
I. Introduction
FACT 1: an uniform genome in almost every cell of an organism
FACT 3: the shape and function of each type of cell are different
FACT 2: the proteome in each type of cell is different
I. Introduction
the actions and properties of each cell type are determined by the proteins it contains
FACT 1: an uniform genome in almost every cell of an organism
transcription of different genes largely determines the actions and properties of cells
FACT 3: the shape and function of each type of cell are different
FACT 2: the proteome in each type of cell is different
I. Introduction
the types and amounts of the various proteins in a cell
the concentration of mRNA and the frequency at which the mRNA is translated
which genes are transcribed and their rateof transcription in a particular cell type
TRUTH: the gene is differentially expressed
regulation
same genome in all cells of an organism
regulation
regulation
I. Introduction
Gene Expression Occurs by a Two-Stage Process
Transcription: generates a single-stranded RNA identical in sequence with one of the strands of the duplex DNA Three principal classes of products:
message RNA (mRNA)transfer RNA (tRNA)ribosomal RNA (rRNA)
Principle: complementary base pairing
Translation: converts the nucleotide sequence of an RNA into the sequence of amino acids comprising a protein
each mRNA contains at least one coding region that is related to a protein sequence
II. Transcription
DNA (gene)
RNA polymerase
Regulatory Proteins
Key Players
promoter
A
startpoint terminator
Transcription Unit
template
upstream downstream
enhancer
II. Transcription
Primary transcript is the original unmodified RNA product correspondingto a transcription unit.
Promoter is a region of DNA involved in binding of RNA polymerase to initiate transcription.
RNA polymerases are enzymes that synthesize RNA using a DNA template(formally described as DNA-dependent RNA polymerases).
Terminator is a sequence of DNA, represented at the end of the transcript,that causes RNA polymerase to terminate transcription.
Transcription unit is the distance between sites of initiation and termination by RNA polymerase; may include more than one gene.
Key Terms
Transcription in eukaryotic cells is divided into three classes. Each class is transcribed by a different RNA polymerase:
RNA polymerase I:
RNA polymerase II:
RNA polymerase III:
RNA Polymerase
II. Transcription
Transcription in eukaryotic cells is divided into three classes. Each class is transcribed by a different RNA polymerase:
RNA polymerase I: rRNA; resides in the nucleolus
RNA polymerase II: mRNA, snRNA; locates in the nucleoplasm
RNA polymerase III: tRNA and other small RNAs; nucleoplasm
II. Transcription
RNA Polymerase
The promoters for RNA polymerases I and II are (mostly) upstreamof the startpoint, but some promoters for RNA polymerase III lie downstream of the startpoint.
Each promoter contains characteristic sets of short conserved sequences that are recognized by the appropriate class of factors.
RNA polymerases I and III each recognize a relatively restricted setof promoters, and rely upon a small number of accessory factors. Promoters utilized by RNA polymerase II show more variation in sequence, and are modular in design.
Promoter
II. Transcription
Short sequence elements (cis-acting elements): bind by accessory factors (transcription factors)
The regulatory region might exist in the promoters of certain eukaryotic genes.
Location: usually upstream and in the vicinity of the startpoint.
These sites usually are spread out over a region of >200 bp. common: used constitutivelyspecific: usage is regulated; define a particular class of genes
These sites are organized in different combinations
Cis-acting Element
II. Transcription
Enhancer element is a cis-acting sequence that increases the
utilization of (some) eukaryotic promoters. The components of an enhancer resemble those of the promoter.
Involve in initiation, but far from startpoint. Are targets for tissue-specific or temporal regulation. Function in either orientation and in any location (upstream or
downstream) relative to the promoter.
Enhancer
two characteristics:1. the position of the enhancer need not be
fixed.2. it can function in either orientation.
II. Transcription
promoter enhancer
position fixed variable
action direction one way either orientation
the density of regulatory elements sparse Heavy (closed packed)
redundancy in function no yes
cooperativity between the binding of factors
sequential great
The Difference between Promoter and Enhancer
The distinction between promoters and enhancers is operational, rather than imply a fundamental difference in mechanism
II. Transcription
Most Eukaryotic Genes Are Regulated by Multiple Transcription-Control Elements
(a) Genes of multicellular organisms contain both promoter-proximal elements and enhancersas well as a TATA box or other promoter element. Enhancers may be either upstream or downstream and as far away as 50 kb from the transcription start site. In some cases, promoter-proximal elements occur downstream from the start site as well. (b) Most yeast genes contain only one regulatory region, called an upstream activating sequence (UAS), and a TATA box, which is ≈90 base pairs upstream from the start site.
II. Transcription
Fact: Regulatory elements in eukaryotic DNA often are many kilobases from start sites
Finding Regulatory Element in Eukaryotic DNA
II. Transcription
Transcription Factor
Any protein that is needed for the initiation of transcription, but which is not itself part of RNA polymerase, is defined as a transcription factor.
binds to DNA (trans-acting factor): recognize cis-acting elements
interacts with other protein: recognize RNA pol, or another factor
The common mode of regulation of eukaryotic transcription is positive: a transcription factor is provided under tissue-specific control to activate a promoter or set of promoters that contain a common target sequence. Regulation by specific repression of a target promoter is less common.
II. Transcription
Accessory factors are needed for initiation, principally
responsible for recognizing the promoter.
Interaction with DNA, RNA polymerase, and/or another
factors.
Three groups:
1. General factors
2. Upstream factors
3. Inducible factors
Another name: accessory factor
II. Transcription
general factors: required for the mechanics of initiating RNA synthesis at all promoters; form a complex surrounding the startpoint with RNA pol, and determine the site of initiation.
basal transcription apparatus (pol + GF)
upstream factors: DNA-binding proteins that recognize specific short consensus elements located upstream of the startpoint. not regulated; ubiquitous; act upon any promoter that contains the appropriate binding site on DNA.
inducible factors: function in the same general way as the upstream factors. have a regulatory role: control transcription patterns in time and space
Accessory Factors
II. Transcription
1. On the genome Which gene(s) to be transcribed? Basic events: Protein binding and/or modification 2. On a specific gene If the gene can be transcribed successfully?
3. On a transcript If the transcript could be correctly spliced? If the transcript could be correctly edited?
Regulation Levels
Key determinant: Cell Signaling!
III. Regulation of transcription
Potential regulation points
Activation of gene structure↓
Initiation of transcription↓
Processing the transcript↓
Termination of transcription↓
Transport to cytoplasm
the overwhelming majority of regulatory events occur at the initiation of transcription
III. Regulation of transcription
5 potential control points:
“Active” Structure
Major Control Point
Alternative Splicing
Regulatory Proteins
the overwhelming majority of regulatory events occur at the initiation of transcription
Key player: regulatory transcription factors
Two questions:1. How does the transcription factor identify its group of target genes?2. How is the activity of the transcription factor itself regulated in response to intrinsic or extrinsic signals?
III. Regulation of transcription
Answer to question 1
The genes share common response element
Structure feature: contain short consensus sequence
Examples:
HSE: heat shock response element; recognized by HSTF
GRE: glucocorticoid response element
SRE: serum response element
MRE: metal response element
III. Regulation of transcription
Regulatory region in MT gene
BLE: basal level element; TRE: TPA response element
General Principle: any one of several different elements, located in either anenhancer or promoter, can independently activate the gene.
III. Regulation of transcription
? = MTF-1
Answer to question 2
Signal transduction
Key events:
1. Protein synthesis
2. Protein modification
3. Ligand binding
4. Protein cleavage
5. Inhibitor release
6. Mutation
III. Regulation of transcription
The activity of a regulatory transcription factor may be controlled by synthesis of protein, covalent modificationof protein, ligand binding, or binding of inhibitors that sequester the protein or affectits ability to bind to DNA.
Reg
ula
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Mod
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ran
scri
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on
Fac
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mutations of the transcriptionfactors give rise to factors thatinappropriately activate, or prevent activation, of
transcription
III. Regulation of transcription
Eukaryotic transcriptional control operates at three levelsduring the stage of initiation
1. changes in chromatin structure directed by activators and repressors
2. modulation of the levels of activators and repressors(gene expression)
3. change the activities of activators and repressors
III. Regulation of transcription
Gene differential expression
IV. RNA Processing
INTRODUCTION
Facts:
1. Genes are interrupted, and mRNAs are uninterrupted
2. The primary transcript has the same organization as the gene
3. Most mRNAs have 5’ cap and 3’ poly(A) tail
4. Heterogeneous nuclear RNAs (hnRNA) exist in the nucleus
5. RNA contains rare bases
Mechanism:
RNA splicing: remove intron
RNA modification: 5’ capping, 3’ polyadenylation, base modification
INTRODUCTION
The initial primary transcript synthesized by RNA polymerase IIundergoes several processing steps before a functional mRNA is produced:
5’ capping 3’ cleavage/polyadenylation RNA splicing
RNA splicing is the process of excising the sequences in RNA that correspond to introns, so that the sequences corresponding to exonsare connected into a continuous mRNA.
IV. RNA Processing
Overview of mRNA Processing in Eukaryotes
The poly(A) tail: ~250 A in mammals, ~150 in insects, ~100 in yeasts. For short primary transcripts with few introns, polyadenylation, cleavage, and splicing usually follows termination. For large genes with multiple introns, introns often are spliced out of the nascentRNA before transcription of the gene is complete.
IV. RNA Processing
Th
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pli
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The splicing snRNPs associate with the pre-mRNA and with eachother in an ordered sequence to form spliceosome
ATP is needed to provide theenergy necessary for rearrangements of the spliceosome structure
IV. RNA Processing
Alternative splicing
Mechanisms:• use of different startpoints or termination sequences• a single primary transcript is spliced in more than one
way, and internal exons are substituted, added, or deleted
Definition: a single gene gives rise to more than one mRNA sequence
Key:what controls the use of such alternative pathways?Proteins? ncRNA?
IV. RNA Processing
Alternative splicing
Mechanisms:• use of different startpoints or termination sequences• a single primary transcript is spliced in more than one
way, and internal exons are substituted, added, or deleted
Definition: a single gene gives rise to more than one mRNA sequence
Key:what controls the use of such alternative pathways?Protein(s)!
IV. RNA Processing
The Troponin (肌钙蛋白) T (muscle protein) pre-mRNA is alternatively spliced to give rise to
64 different isoforms of the protein
Constitutively spliced exons (exons 1-3, 9-15, and 18)
Mutually exclusive exons (exons 16 and 17)
Alternatively spliced exons (exons 4-8)
Exons 4-8 are spliced in every possible waygiving rise to 32 different possibilities
Exons 16 and 17, which are mutually exclusive,double the possibilities; hence 64 isoforms
IV. RNA Processing
Trans-(intermolecular) splicing
Splicing is usually cis-reaction (intramolecular), but trans-(intermolecular) splicing have been found (very rare). These reactions probably occur by splicesome formation with the appropriate site sequences on each molecule.
trypanosomes and euglenoids: all the mRNAsCaenorhabditis elegans: 10-15% of the mRNAsHuman?
IV. RNA Processing
Initiation of Protein Synthesis
V. Initiation of Protein Synthesis
Critical event:begin protein synthesis at the start codon, thereby setting the stagefor the correct in-frame translation of the entire mRNA.
Main mechanisms:Base pairing between mRNA and rRNABase pairing between mRNA and tRNAMet-tRNAi
Met can only bind at the P site to begin synthesis
Participants: Met-tRNAi
Met
mRNA IFs small subunit large subunit
Protein translation
Two types of methionine tRNA are found in all cells
same aminoacyl-tRNA synthetase (MetRS) charges both tRNAs with methionine
V. Initiation of Protein Synthesis
Model of protein synthesis on circular polysomes and recycling of ribosomal subunits
PABI and eIF4 (4G and 4E) can interact on mRNA to circularize the molecule
V. Initiation of Protein Synthesis
The nascent polypeptide chain must undergo folding and, in many cases, chemical modification and cleavage to generate the final protein
Folding:Theoretically: any polypeptide chain containing n residues could, in principle, fold into 8n conformations. Fact: adopt a single conformation (native state)
a single, energetically favorable conformation Mechanism: the amino acid sequence provides the information for protein folding
Protein Maturation
Modification: N terminal C terminal Certain sites btw N and C terminus
VI. Protein Processing
Nearly every protein in a cell is chemically altered after its synthesis in a ribosome, thus alter its activity, life span, or cellular location of proteins,
depending on the nature of the alteration.
Two categories:chemical modification involves the linkage of a chemical group to the terminal amino or carboxyl groups or to reactive groups in the side chains of internal residues may be reversibleProcessing involves the removal of peptide segments and generally is irreversible
Protein Alteration
VI. Protein Processing
The internal residues in proteins can be modified by attachment of a variety ofchemical groups to their side chains:phosphorylation (Ser, Thr, Tyr) glycosylation (Asp, Ser, Thr)ubiquitinationothers
Examples of modified internal residuesproduced by hydroxylation, methylation, and carboxylation
Protein Modification
VI. Protein Processing
Protein Cleavage
most common form:
residues are removed from the C- or N-terminus of a polypeptide
by cleavage of the peptide bond in a reaction catalyzed by
proteases.
Proteolytic cleavage is a common mechanism of activation or
inactivation
Proteolysis also generates active peptide hormones
EGF; insulin
VI. Protein Processing
Protein Degradation
Two Pathways
extracellular: digestive proteases
intracellular
lysosomes
cytosolic mechanisms
The ubiquitin-mediated pathway is the best-understood cytosolic pathway.In ubiquitinating enzyme complex, different conjugating enzymes recognize different degradation signals in target proteins.
ubiquitin-conjugating enzyme E1: Arg-X-X-Leu-Gly-X-Ile-Gly-Asxcertain residues at the N-terminus favor rapid ubiquitination
VI. Protein Processing