Transcription in Prokaryotes

Transcriptional Control

DNA

RNA

protein

Environmental change

Turn gene(s) on/off

Proteins to deal with new environment

Very important to:1. express genes when needed2. repress genes when not needed3. Conserve energy resources; avoid expressing unnecessary/detrimental genes

Transcriptional Control

DNA

RNA

protein

TranscriptionInitiationElongationTermination

ProcessingCappingSplicingPolyadenylationTurnover

Translation

Protein processing

Many places for control

Prokaryotic Transcription

OperonsGroups of related genes transcribed by the same promoter

Polycistronic RNA

Multiple genes transcribed as ONE TRANSCRIPT

No nucleus, so transcription and translation can occur simultaneously

RNA Structure

Contain ribose instead of deoxyribose

Bases are A,G,C,U, Uracil pairs with adenine Small chemical difference from

DNA, but large structural differences

Single stranded helix Ability to fold into 3D shapes - can

be functional

RNA Structures Vary

RNA more like proteins than DNA:structured domains connected by more flexible domains, leading to different functions

e.g. ribozymes – catalytic RNA

RNA synthesis •

•

RNAP binds, melts DNA

Nucleosides added 5’ 3’

Types of RNA

Messenger RNA (mRNA) – genes that encode proteins

Ribosomal RNA (rRNA) – form the core of ribosomes

Transfer RNA (tRNA) – adaptors that link amino acids to mRNA during translation

Small regulatory RNA – also called non-coding RNA

Transcriptional Control

TranscriptionInitiationElongationTermination

ProcessingCappingSplicingPolyadenylationTurnover

Translation

Protein processing

Control of initiation usually most important.

Initiation

RNA polymerase Transcription factors Promoter DNA

RNAP binding sites Operator – repressor binding Other TF binding sites

Start site of txn is +1

α α β β’σ

Initiation

RNA polymerase 4 core subunits Sigma factor (σ)– determines promoter specificity Core + σ = holoenzyme Binds promoter sequence Catalyzes “open complex” and

transcription of DNA to RNA

RNAP binds specific promoter sequences

Sigma factors recognize consensus-10 and -35 sequences

RNA polymerase promoters

TTGACA TATAAT

Deviation from consensus -10 , -35 sequence leads to weaker gene expression

Bacterial sigma factors

Sigma factors are “transcription factors” Different sigma factors bind RNAP and recognize

specific -10 ,-35 sequences Helps melt DNA to expose transcriptional start

site Most bacteria have major and alternate sigma

factors Promote broad changes in gene expression

E. coli 7 sigma factors B. subtilis 18 sigma factors

Generally, bacteria that live in more varied environments have more sigma factors

Sigma factors

E. coli can choose between 7 sigma factors and about 350 transcription factors to fine tune its transcriptional output

An Rev Micro Vol. 57: 441-466 T. M. Gruber

Sigma subunit Type of gene controlled # of genes controlled

RpoD Growth/housekeeping ~1000

RpoN N2; stress response ~15

RpoS Stationary phase, virulence ~100

RpoH Heat shock ~40

RpoF Flagella-chemotaxis ~40

RpoE ? ~5

FecI Ferric citrate transport ~5

Extreme heat shock, unfolded proteins

s70

s54

sS

sS

sF

s32

What regulates sigma factors

Number of copies per cell (σ70 more than alternate)

Anti-sigma factors (bind/sequester sigma factors)

Levels of effector molecules Transcription factors

Bacterial RNAP numbers

In log-phase E. coli: ~4000 genes ~2000 core RNA polymerase molecules ~2/3 (1300) are active at a time ~1/3 (650) can bind σ subunits.

Competition of σ for core determines much of a cell’s protein content.

Lac operon control

• Repressor binding prevents RNAP binding promoter

• An activating transcription factor found to be required for full lac operon expression: CAP (or Crp)

lac operon – activator and repressor

CAP = catabolite activator protein

CRP = cAMP receptorprotein

Activating transcription factors

Helix-turn-helix (HTH) bind major groove

of DNA HTH one of

many TF motifs

Crp dimer w/ DNA

Cofactor binding alters conformation

Crp binds cAMP, induces allosteric changes glucose

cAMP

Crp

lac operon

no mRNA

cAMP

Crp

glucose

mRNA

Cooperative binding of Crp and RNAP

Binds more stably than either protein alone

Enhancers• activating regions not necessarily close to RNAP binding site

• NtrC required for RNAP to form open complex

NtrC example:

• NtrC activated by P

• P NtrC binds DNA, forms loop that folds back onto RNAP, initiating transcription• signature of sigma 54

Bacterial promoters

Most bacterial promoters have –35 and –10 elements

Some have UP element Some lack –35 element, but have extended

–10 region

-35 element -10 element (Pribnow box)UP element

pre –10 element

+1

Transcription start

+1

E. coli RNA polymerase composed of 5 subunits:

•Subunits: b, b’, a(2), , and s

•Core enzyme: b, b’, a(2), •Holoenzyme : b, b’, , a(2) and s

•The s subunits give specificity for site of initiation- promoter

Subunit structure of bacterial RNA polymerase

’NTDCTD

NTDCTD

sDNA

Holoenzyme-b’ba2s. Functions in initiation.

Core enzyme-b’ba2. Functions in elongation.

160 kDa

150 kDa

40 kDa

The 3D structure of bacterial RNA polymerase holoenzyme

N-term s1

Inhibition

s2

-10 binding

s3

-10 binding

s4

-35 binding

s factor domains :

s3

The s factors s factors are required for promoter recognition and

transcription initiation in prokaryotes s factors have analogous function as general

transcription factors in eukaryotes A variety of s factors exist in E.coli For expression from most promoters s70 is required For expression from some bacterial promoters one

of other s subunits is needed instead s70 is essential for cell growth in all conditions, while

other sigmas are required for special events, like nitrogen regulation (s54), response to heat shock (s32), sporulation, etc

s

RNA pol

Holoenzyme

Promoter region

Closed complex

Open complex

Promoter escape

Elongation

mRNA s release

-35 -10

The overview of s factor function

The promoter specificity of some s factors in E.coli

s70 TTGACA – 17 bp – TATAATN3-6-A -35 -10 +1

s32 CTTGAAA – 16 bp – CCCCATNTN3-10-T/A -35 -10 +1

s54 GG – N12 – GC/T – 12bp – A -24 -12 +1

The UP element

UP element is an AT rich motif present in some strong (e.g. rRNA) promoters

UP element interacts directly with C-terminal domain of RNA polymerase asubunits

-35 -10 UP +1

s4 s2-3sRNAPa NTD

a CTD

RNAP

Constitutive and inducible promoters

Certain genes are transcribed at all times and circumstances

-Examples – tRNAs, rRNAs, ribosomal proteins, RNA polymerase

-Promoters of those genes are called constitutive Most genes, however, need to be transcribed

only under certain circumstances or periods in cell life cycles

-The promoters of those genes are called inducible and they are subject to up- and down- regulation

Regulation at promoters

Promoters can be regulated by repression and/or activation

Many s70 promoters are controlled both by repression and activation, whereas, for example s54 promoters are controled solely by activation

Cartoon of the transcription cycle

Mechanisms of repression

Repression by steric hindrance Inhibition of transition to open complex Inhibition of promoter clearance Anti-activation Anti-sigma factors

e) Anti-sigma factors An anti-s factor is defined by the ability to prevent

its cognate s factor to compete for core RNA polymerase

Mostly used for s factors, other than s70, for example in life cycle regulation (sporulation, etc)

Some bacteriophages use their own anti-s factors to prevent expression of cellular proteins

-10 -35

RNAP

s

-10 -35

RNAP

santi-s

d) Anti-activation Repressor molecule removes the

activator

weak promoter +1

weak promoter ABS +1

ABS

RNA pol - s

Activator

RNA pol - sActivator

Repressor

Activator binding sequence

Two examples of steric hindrance

Trp repressor Lac repressor

The tryptophan repressor The trp repressor controls the operon for the

synthesis of L-tryptophan in E.coli by a simple negative feedback loop

When enough tryptophane (blue dots) is made, it binds to repressor, which now is able to bind to promoter and block RNA polymerase binding

In the absence of tryptophane the trp repressor (red blob) shows no affinity to promoter (black box) and the RNA polymerase (yellow blob) transcribes the operon

The lac promoter

Lac promoter is widely used in artifical plasmids, designed for protein production

For practical purposes it is easier to use non-hydrolyzable lactose analog – IPTG (isopropyl-b-thiogalactoside) instead of native lactose

A cartoon, ilustrating events upon IPTG binding to lac repressor

As IPTG binds, the DNA binding domains scissor apart

(IPTG)

Mechanisms of activation

a) Regulated recruitment b) Polymerase activation c) Promoter activation

a) Regulated recruitment

Activator “extends” the binding site for RNA polymerase

weak promoter +1 ABS

RNA pol - s

Activator

strong affinity weak affinity

strong or weak affinity

Catabolite Activator Protein: CAP

Activates transcription from more than 150 promoters in E.coli

Upon activation by cAMP (cyclic Adenosine MonoPhosphate), CAP binds to promoter and helps RNAP-s to bind as well

All CAP–dependent promoters have weak –35 sequence, so that RNAP-s is unable to bind the promoter without CAP assistance

Models for Class I and Class II promoter activation

Busby and Ebright, 2000, J. Mol. Biol. 293:199-213

Class I CAP binding sites can be from –62 to –103. CAP interacts with the carboxy terminal domain of the RNAP a-subunit (aCTD)

Class II CAP binding sites usually overlap the –35. CAP interacts with the aCTD, aNTD (N-terminal domain), and the s factor

Model for Class III promoter activation

Activation of Class III promoters requires binding of at least two CAP dimers or at least one CAP dimer and one regulation-specific activator

Interactions can be similar to those of ClassI and/or ClassII promoters, except that each aCTD subunit is making different interactions

AraC – repressor and activator of arabinose

promoter

RNAP-s

RNAP-s

Transcription

+ arabinose ( )

AraC

promoter

DNA binding domain of AraC

RNAP-s54 activation

RNAP-s54 open complex formation requires ATP hydrolysis Activator protein with ATP-ase activity binds to “enhancer”

site about 160 bp upstream from –24 sequence. DNA then gets looped and activator interacts with RNAP-s54 resulting in the open bubble formation upon ATP hydrolysis

s54

s54

ATP ATP+Pi

c) Example of promoter activation: MerR activator family

MerR is an activator that controls genes involved in the response to mercury poisoning

Other MerR family activators (CueR, BmrR, etc) respond to a variety of different toxic compounds such as other heavy metal atoms or drugs

In MerR activated promoters, -10 and –35 regions are separated by 19bp instead of optimal 17bp

DNA-protein interaction assays

Electrophoretic mobility shift assay (EMSA)

DNase I Footprinting

Chromatin immunoprecipitation (ChIP)

EMSA

Radiolabel promoter sequence

Incubate one sample with cell lysates or purified protein and the other without

TF will bind promoter sequence

Run DNA-protein mixture on polyacrylamide gel and visualize w/ audoradiography Free probe

TF-bound probe

EMSA

CovR PcylE

CovR DNA binding proteinBinds to cylE promoter Recognition sequence ‘TATTTTAAT’

DNase I Footprinting

Method to determine where a protein binds a DNA sequence

DNase I footprint

1 -- DNA sequence ladder2 -- DNA sequence ladder3 -- No protein4 -- (+) RNA polymerase5 -- (+) lac repressor

ChIP

Crosslink proteins bound to DNA

Immunoprecipitate lysate for specific transcription factor, RNAP, etc

Analyze DNA bound to protein by PCR

Transcriptional Control

TranscriptionInitiationElongationTermination

ProcessingCappingSplicingPolyadenylationTurnover

Translation

Protein processing

Transcriptional Termination

Bacteria need to end transcription at the end of the gene

2 principle mechanisms of termination in bacteria: Rho-independent (more common) Rho-dependent

Rho-independent termination

• Termination sequence has 2 features:Series of U residuesGC-rich self-complimenting region• GC-rich sequences bind forming stem-loop• Stem-loop causes RNAP to pause• U residues unstable, permit release of RNA chain

Rho-dependent termination Rho is hexameric

protein 70-80 base segment of

RNA wraps around Rho has ATPase activity,

moves along RNA until site of RNAP, unwinds DNA/RNA hybrid

Termination seems to depend on Rho’s ability to “catch up” to RNAP

No obvious sequence similarities, relatively rare

Transcriptional attenuation Attenuator site = DNA sequence where RNAP

chooses between continuing transcription and termination

trp operon 4 RNA regions

for basepairing 2 pairs w/ 1 or 3 3 pairs w/ 2 or 4 Concentration of Trp-tRNATrp determines fate of attenuation At high Trp conc, transcription stops via Rho-independent

Anti-terminationλ phage encode protein that prevents termination

Two Component Systems

Two Component Systems

‘Histidine kinase’ senses environmental changes- autophosphorylates at conserved histidine residue

Response regulator is phosphorylated by activated sensor kinase at conserved aspartate- activates or represses transcription/function

Way for bacteria to sense environmental changes and alter gene expression

Quorum Sensing

Bacteria produce and secrete chemical signal molecules (autoinducers)

Concentration of molecules increases with increasing bacterial density

When critical threshold concentration of molecule is reached, bacteria alter gene expression

Way for communities of bacteria to “talk” to each other

Quorum Sensing in Vibrio fischeri

• at high cell density, V. fischeri express genes for bioluminescence

• LuxI produces autoinducer acyl-homoserine lactone

• AHL diffuses outside of cell

• when AHL reaches critical concentration, it binds LuxR

• activated LuxR bound AHL activates transcription of luminescence genes

Transcription termination

In prokaryotes two types of transcription termination occur – rho indepedent termination and rho dependent termination

In rho independent case, the termination is achieved by a secondary structure of mRNA – RNA stem-loop, followed by an AU rich region

A rho protein is required for rho-dependent termination

Rho independent termination

Attenuation

Regulation of transcription by the behavior of ribosomes

Observed in bacteria, where transcription and translation are tightly coupled

Translation of a mRNA can occur as the mRNA is being synthesized

Attenuation in trp operon

Rho dependent terminationAs polymerase transcribes away from the promoter, rho factor binds to RNA and follows the polymerase

When polymerase reaches some sort of pause site, rho factor catches up with polymerase and unwinds the DNA-RNA hybrid, resulting in release of polymerase

Anti-termination