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Biochemistry I Fall Term, 2004 October 20, 2004

Lecture 20: Transcription Regulation

Assigned reading in Campbell: Chapter 10 (Section 10.6 omitted)

Key Terms:

lac Repressor & operator

CAP-cAMP activation

Closed & open promoter complexes

Initiation frequency

CREB, CRE, and CBP

Leucine zipper motif (bZIP)

Helix-turn-helix domain (HTH)

Zinc finger domain

Links:

(I) Review Quiz on Lecture 20 concepts

(S) Protein-Nucleic Acid Complexes A page of JPEG images to accompany this lecture.

Structural Motifs in DNA-Binding Proteins (Campbell, Section 10.5):

(S) CREB-DNA Complex: Human bZIP Protein. (Chime)

(S)E. coliCAP-cAMP-DNA Complex: Helix-turn-Helix Activator. (Chime)

(S) Zif268-DNA Complex: Human Zinc-Finger Protein. (Chime)

3. Control of Transcription in Prokaryotes.

A. Expanded diagram of the lac promoter-operator region (PO).

Binding sites for the proteins are indicated with arrows. mRNA synthesis starts within the

PO region and extends into theZ, Y, andA genes.

B. Mechanism of RNA chain initiation.

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R = RNA polymerase holoenzyme P = promoter

RPc = "closed complex" RPo = "open complex"

KB= binding constant for RPc (M-1 ) kf= rate constant for RPo formation from RPc (sec-1).

Using the above scheme, measurements of initiation reactions have provided quantitative

estimates of KB and kf that are analogous to the enzyme kinetic parameters, KM and Vmax in theMichaelis-Menten equation. The following are conclusions from that work:

1. Both KB and kfdiffer for different promoters.

a. KB : the range is from 106 M-1 to 109 M-1.

b. kf: the range is from 10-4 sec-1 to 10-1 sec-1.

c. Initiation frequencies vary from 10 RNA chains/min to

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4. Transcription in Eukaryotes

A. RNA polymerase II synthesizes mRNA

1. Several subunits are homologous to bacterial RNA polymerases

2. Additional subunits and modifications, i.e not found in bacteria

3. The polymerization reaction is identical in all species.

B. A (large) number of additional initiation factors (GTFs) are required.

e.g. TFIID-TBP and the TFIID-TAFIIs

5. Regulation of Eukaryotic Transcription

A. Most promoters are inaccessible to RNA Pol II.

1. Chromatin = DNA:histones: other proteins = 1:1:1.

2. Proteins remodel chromatin to make it accessible.

B. Histone modification can expose promoters (and other sites).

1. Acetylation of ~20 Lys residues on the histone N-terminal tails.

NH3

+NH

CH3

O

+ AcetylCoA

2. Phosphorylation (Ser) and methylation (Lys & Arg) also contribute.

C. CREB-CRE-CBP: A specific activation pathway (signal transduction cascade)

1. Hormone binding to a cell-surface-receptor causes:

2. Increase in cAMP ("second messenger") inside the cells, which

3. Activates protein kinase A (PKA), which

4. Phosphorylates CREB (cAMP response element binding protein).

5. CREB binds the CRE site (cAMP response element) at several promoters

6. CBP bridges between CREB and RNA Pol II (basal complex).

7. Transcription is turned on.

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D. CREB and other activators have modular architectures:

1. Activation domain DNA binding domain Protein interaction domains

2. Examples of DNA binding domains (DBDs):

a. bZIP: CREB and Fos-Jun

b. Zinc finger: Zif268

c. Helix-turn-helix: CAP

6. Forces and Functional Groups Involved in DNA Recognition.

A. Electrostatic bonding to the backbone.

1. Side chains of Lys and Arg to phosphates.

2. Release of M+ (and H2O) favors binding (+S).

B. Hydrogen bonding to the polar edges of the bases and sugars.

1. Side chains of Arg, Asn, Gln; e.g. Gln-Adenine & Arg-Guanine Fig. 10.24

2. Main chain, C=O and NH groups.

3. "Bridging H2O" can also participate.

C. Stacking (and intercalation) of Phe, Trp, and Tyr side chains.

D. van der Waals (hydrophobic) interaction with the 5-methyl of T.

Except for the stacking interaction, this list is the same set of forces and functional groups

that we have seen in enzyme-substrate, antibody-antigen, and other protein-binding

interactions.

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