Transcription regulation in prokaryotes
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Transcription regulation in prokaryotes. Background: major and minor grooves of DNA. Available interactions in minor and major grooves. Schematic view of available chemical groups in minor and major grooves. In major groove all 4 nucleotides can be differentiated by a binding protein

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Transcription regulation in prokaryotes

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Transcription regulation in prokaryotes

Transcription regulation in prokaryotes


Background major and minor grooves of dna

Background: major and minor grooves of DNA


Available interactions in minor and major grooves

Available interactions in minor and major grooves


Schematic view of available chemical groups in minor and major grooves

Schematic view of available chemical groups in minor and major grooves

  • In major groove all 4 nucleotides can be differentiated by a binding protein

  • In minor groove only AT or GC base pairs can be differentiated

  • The minor groove is also much narrower, which makes it less accessible for binding proteins

  • As a consequence, most sequence specific DNA binding proteins bind to major groove


Helix turn helix dna binding motif

Helix-turn-helix DNA binding motif

  • Helix-turn-helix motif is the most common DNA-binding motif in prokaryotes, present in many transcription repressors and activators

  • One of the helices, DNA recognition helix, gets inserted in the major groove of DNA

  • Helix-turn-helix proteins are often dimeric, with two recognition helices recognizing two adjacent DNA sequences

  • Why dimeric?

  • Dimer binds to DNA stronger than monomer

  • By changing the relative positions of monomers, the dimer activity can be easily turned on and off


A common principle to activate or inactivate dimeric helix turn helix proteins

A common principle to activate or inactivate dimeric helix-turn-helix proteins

  • Ligand changes the position of DNA binding a helices, so they do not bind DNA any more

  • Or the opposite – ligand changes the position of helices, so they do bind to DNA

(ligand)


Transcription regulation in prokaryotes

The main features of interactions between DNA and the helix-turn-helix motif in DNA-binding proteins

  • 1. Sequence unspecific interactions contribute to overall stability of complex, but do not differentiate the ound DNA sequence

  • 2. Sequence specific interactions determine, with which regions of DNA the interaction will occur


Bacterial promoters

Bacterial promoters

Transcription start

+1

UP element

  • 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)

+1

pre –10 element


The s factors

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


The overview of s factor function

RNA pol

s

Holoenzyme

Promoter region

-35 -10

Closed complex

Open complex

Promoter escape

Elongation

mRNA

s release

The overview of s factor function


The promoter specificity of some s factors in e coli

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


Some s 70 promoters in e coli

Some s70 promoters in E.coli

The –35 and –10 sequences of individual s factors are conserved (yellow boxes)

The spacer sequence between –35 and –10 is not conserved, but the spacer length is 171 bp


The 3d structure of bacterial rna polymerase holoenzyme

The 3D structure of bacterial RNA polymerase holoenzyme

s3

s factor domains :

N-term s1

Inhibition

s2

-10 binding

s3

-10 binding

s4

-35 binding


The up element

The UP element

RNAP

RNAP

a NTD

  • 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 a subunits

s

a CTD

s4

s2-3

UP

-35

-10

+1


Constitutive and inducible promoters

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

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


Mechanisms of repression

Mechanisms of repression

  • Repression by steric hindrance

  • Inhibition of transition to open complex

  • Inhibition of promoter clearance

  • Anti-activation

  • Anti-sigma factors


A repression by steric hindrance most cases

a) Repression by steric hindrance (most cases)

  • Examples: Trp repressor, lac repressor


B inhibition of transition to open complex

b) Inhibition of transition to open complex

  • RNA Polymerase – s complex (RNAP-s) can bind to promoter, but transition to open complex is blocked


C inhibition of promoter clearance

c) Inhibition of promoter clearance

  • The transcription bubble can be formed, but further RNAP-s movement is blocked


E anti sigma factors

e) Anti-sigma factors

  • An anti-s factor is defined by the ability toprevent 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

RNAP

RNAP

anti-s

s

s

-10 -35

-10 -35


D anti activation

d) Anti-activation

  • Repressor molecule removes the activator

RNA pol - s

Activator

ABS

weak promoter

+1

Activator binding sequence

Activator

RNA pol - s

Repressor

ABS

weak promoter

+1


Two examples of steric hindrance

Two examples of steric hindrance

  • Trp repressor

  • Lac repressor


The tryptophan repressor

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

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

The tryptophan repressor

  • The trp repressor controls the operon for the synthesis of L-tryptophan in E.coli by a simple negative feedback loop


The 3 d structure of trp repressor

The 3-D structure of trp repressor


Transcription regulation in prokaryotes

The conformational change upon binding tryptophan molecules induces a conformational change in trp repressor


The lac promoter

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


The structure of lac repressor monomer

The structure of lac repressor monomer

Hinge helix

DNA binding domain

Core N subdomain

Inducer binding pocket

Core C subdomain

Tetramerization helix


Functional lac repressor is a homotetramer

Functional lac repressor is a homotetramer

  • Each dimer binds to a distinct DNA sequence at –82 and +11 respective to transcription start site

  • This results in DNA looping, preventing the DNA polymerase from binding to –35 and –10 sequences

+11

-82

-35

-10

-82

+11

lac repressor


The lac repressor binds both to major and minor grooves of dna

The lac repressor binds both to major and minor grooves of DNA


A cartoon ilustrating events upon iptg binding to lac repressor

A cartoon, ilustrating events upon IPTG binding to lac repressor

(IPTG)

As IPTG binds, the DNA binding domains scissor apart


Mechanisms of activation

Mechanisms of activation

  • a) Regulated recruitment

  • b) Polymerase activation

  • c) Promoter activation


A regulated recruitment

a) Regulated recruitment

  • Activator “extends” the binding site for RNA polymerase

strong or weak affinity

RNA pol - s

Activator

ABS

weak promoter

+1

strong affinity

weak affinity


Catabolite activator protein cap

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


Transcription regulation in prokaryotes

Models for Class I and Class II promoter activation

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

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


Model for class iii promoter activation

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


Transcription regulation in prokaryotes

80º

3-D structure of CAP-cAMP-DNA complex

Binding of CAP causes DNA bending by 80º

C

C

Cyclic AMP binds to the N-terminal domain and causes the two long “C” helices to reorient and move the DNA binding domains apart, so that CAP can bind to DNA


Cap activates lac operon

CAP activates lac operon


Arac repressor and activator of arabinose promoter

DNA binding domain of AraC

AraC

RNAP-s

promoter

+ arabinose ( )

Transcription

RNAP-s

AraC – repressor and activator of arabinose promoter


Structure of arac dimers in presence a and absence b of arabinose

Structure of AraC dimers in presence (A) and absence (B) of arabinose

  • Without arabinose, the monomers interact with the b barrel domains. An important interaction is stacking of Tyr31 of one subunit on Trp 95 of the other

  • Arabinose binds in close proximity to Trp95, making the stacking interaction impossible Monomers associate in a different way – 4 helix bundle interactions by the helical domains


B polymerase activation

b) Polymerase activation

  • This works for s54 promoters

  • RNAP-s54 forms a stable complex with DNA, but needs to be activated to form an open complex


Rnap s 54 activation

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

ATP+Pi

ATP

s54

s54


C example of promoter activation merr activator family

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


The bmrr dna drug complex

The BmrR-DNA-drug complex

  • BmrR binds to a variety of toxic compounds, including tetraphenylphosphine (TPP)

  • Both TPP bound and unbound forms of BmrR bind to promoter, but only TPP bound form induces transcription


Transcription regulation in prokaryotes

  • In 19-bp spacer variant, -35 and –10 binding regions not only are too far from each other, but also on the opposite sides of double helix

  • In BmrR-TPP bound vairiant, DNA double helix is underwound, so that –10 and –35 regions are at the same distance as in regular 17-bp spacer


Transcription termination

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

Rho independent termination


Attenuation

Attenuation

  • Regulation of transcriptionby thebehavior of ribosomes

  • Observed in bacteria, wheretranscription and translation aretightly coupled

  • Translation of a mRNA can occur asthe mRNA is being synthesized


Attenuation in trp operon

Attenuation in trp operon


Rho dependent termination

Rho dependent termination

As 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

Anti-termination


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