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An overview of protein synthesis via transcription and translation

References:

1. Genes VIII, by Lewin, 2004, Oxford.

2. Molecular Biology, by Weaver, 3rd ed.,2004, McGraw-Hill.

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Prokaryotic Gene Expression

Transcription(RNA polymerase)

Translation(Ribosome)

Promoter

Terminator+1-10-35

Ribosome-binding site

Start codon Stop codon

ORFmRNA

Protein

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Transcription in Prokaryotic Cells

RNA polymerase

DNA template

Coding strand

Template strand

promoter;

terminator

RNA pol RNA pol

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Template recognition – Initiation – Elongation - Termination

5’

3’

Stages of transcription

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RNA pol ineubacteria core: 2’ holoenzyme:

core + factor

factor is separated from the core when holoenzyme is subjected to an anion exchange (e.g. phosphocellulose) column

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Yeast RNA polymerase

RNA

DNA

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Promoter recognition.

Promote tight binding of holoenzyme to the promoter.

Loosening non-specific interaction between RNA pol and template.

Stimulates transcription initiation.

Effect of factor

Functions of factor

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converts a loosely b

ound RNA pol in a clo

sed complex to the tig

htly bound pol in the

open promoter compl

exes.

Supercoiled DNA is a

better template for tra

nscription, because it

requires less free ener

gy for the initial meltin

g of DNA.

RNA pol-promoter binding

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Initiation

1. Forming the closed promoter complex

2. Forming the open promoter complex

3. Abortive initiation

4. Promoter clearance

Template recognition

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Sliding along DNA does not occur

How RNA polymerase gets to the promoter?

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+Rifampicin

-Rifampicin

Rif R

Rif S

Sigma cycle

factor can be reused

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DNA region covered by holoenzyme is from -55 to +20; that covered by core enzyme after loss of is from -35 to +20.

RNA Pol-Promoter Interaction

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DNA footprinting

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RNA Pol-Promoter Interaction

Methylation Interference Assay

Bases on either the template or the non-template strand that are more methylated in the filtrate than in the filter-bound DNA are presumably important in polymerase binding to the promoter.

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RNA pol-promoter contact

-9 to +3

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Features of bacterial promoters

Consensus

-10 +1

TTTACA

TTGACA

TTGACA

TTGATA

TTGACA

TATGTT

TTAACT

GATACT

TATAAT

TATGTT

TTGACA TATAAT

18 bp

17 bp

17 bp

17 bp

9 bp

18 bp

7 bp

lactrp

lac

trp

lPL

recA

tacI

-35

>90% of transcription start point is a purine

(16-19 bp) (5-9 bp)

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How many kinds of factors are there in a bacterial cell?

What is the structure of factor?

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Primary factors

(e.g. 70 of E. coli;

43 of B. subtilis )

Alternative factors

Transcription of speci

alized genes

(e.g. 54 of E. coli)

Structure of factors

Free cannot bind to the promoter (The N-terminal region suppresses the DNA-binding region). Only when it is bound with the core, upon which its conformation changes, can binds the promoter.

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Region 1. Present only in primary . The 245 aa existing i

n 70, but not in 43, may be involved in loosening bindi

ng between RNA pol and non-promoter regions.

Region 2. Most highly conserved.

2.1 and 2.2: hydrophobic; binding to pol core.

2.3: involved in DNA melting.

2.4: -helix; recognition of -10 box.

Region 3. Helix-turn helix DNA-binding domain.

Region 4.

4.2: helix-turn-helix loop; binding to -35 box.

70

245 aa deletion

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54 is different from other factors in:

1. The “-35 box” is located 6 bp upstream of the “-10 box”;

2. Sites that are rather distant from the promoter influence its activity (recognized by an enhancer-binding protein);

3. The free form can bind to DNA.

Different factors recognize promoters with different consensus sequences

Primary vs. alternative factors in E. coli

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Sigma-switching model

Temporal control of transcription of B. subtilis phage SPO1

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Other examples :Control of transcription during sporulation in B. subtilisRegulation of glutamine synthetase gene (54)Regulation of the E. coli heat shock genes (32)Stress-resistance genes turned on in the stationary phase (s)

Genetic evidence

Isolation of mutants that are unable to do transcription switch.

Biochemical evidence

Composition analysis of the RNA pol isolated from different stages.

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Functions of RNA pol core

1. To unwind and rewind DNA

2. To hold the separated strand of DNA and RNA

3. To catalyze the addition of ribonucleotides to the

growing RNA chain

4. To adjust the difficulties in processing by cleavin

g the RNA product and restarting RNA synthesis (

with the assistance of some accessory factors, e.

g., GreA and GreB in E. coli)

Elongation

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Recover of RNA polymerase from pausing

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Role of subunit in UP element recognition:

1. Addition of UP to the cor

e promoter increases in vitr

o transcription by RNA pol a

lone.

2. The 94 C-terminal aa are r

equired for UP recognition.

Function of -subunitCore enzyme assembly; Promoter recognition;

Interaction with some regulators.

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UP element: an AT-rich sequence which stimulates trans

cription of the rrnB gene by a factor of 30.

Fis sites: binding sites for Fis, a transcriptional activator

.

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-subunit

Phosphodiester bond formation.

(Confers both the rifampicin- an

d streptolydigin-resistance)

Stabilizing RNA pol-DNA complex

during elongation.

Forms both the salt-sensitive and

salt-resistant contact with the D

NA template.

’-subunit

Most basic subunit.

Strongest DNA-

binding activity.

Forms salt-resistant

contact with the

DNA template.

Function of and ’-subunit

31The strain of unwinding is relaxed by the topoisomerases.

Topology of elongation

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Termination Mechanism

-independent termination(intrinsic terminators)

Requires:

a hairpin loop

a string of Ts following

the hairpin.

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Intrinsic terminators

Stem of hairpin: G-C-rich; 7-20 bp

Loop: 5 bp or up

Distance between hairpin and U-run: 7-9 bp

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Requires a hairpin loop and

factor acts as a homohexamer

each subunit contains

an RNA-binding domain

an ATPase domain.

an RNA helicase (separates

RNA-DNA hybrid)

-dependent terminationHalf of E. coli terminators; most are found in phage genomes

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-dependent terminator

50-90 bases longC-rich/G poor

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Polar effect on transcription of the downstream genes caused by a nonsense mutation

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Negative control:

Positive control:

Control of prokaryotic transcription

Inducible genes

v.s.

Constitutive genes

Repressor

Activator

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Cofactors of the regulators:Repressor

Inducer

Corepressor

ActivatorInducer

Common features of the cofactors:

Highly specific

Not necessarily interact with the target enzyme

Gratuitous inducers (e.g., IPTG)

Allosteric control of the regulator

Other positive control mechanisms:

Substitution of factors

Antitermination

Other means of activation of regulators:

Phosphorylation

Oxidation

394

Operon

A group of contiguous, coordinately controlled genes

Polycistronic mRNA

The first operon discovered (Jacob and Monod, 1961)

The lac operon