62 Gene Regulation

8/12/2019 62 Gene Regulation

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6.2.4 Termination of Transcription

1. Intrinsic (factor-independent) termination: abouthalf of the transcription units in E.coli

2. Factor-dependent termination (Rho factor): the

other half

3. Damage-dependent termination (Mfd factor)

Three processes destabilize the elongation

complex and release the transcript:


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The factor-independent (intrinsic)

has two essential components:

1. A GC-rich hairpin that forms in the emergingtranscript and is closed about 9 nucleotides

upstream of the RNA release site

2. An adjacent U-rich segment: the weakness

of the rU/dA hybrid is thought to facilitate its

unwinding and dissociation


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Factor-Independent Terminator


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Model of Rho-Independent

Termination


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Forms hexamers carrying six RNA-binding sites

and six ATP-binding sites

Binds to a loading site (rut = rho utilization):~ 70 nucleotides long contains a significant

number of cytosine residues and no secondarystructure

Formation of mRNA-Rho complex leads to

activation of Rho ATPase facilitating

translocation in a 5' 3' direction

Rho-Dependent Termination


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Rho can terminate transcription frequently in

operons

Rho can attenuate transcription conditionally

at the beginning of operons

Rho can attenuate transcription even within

open reading frames when mRNA is

uncovered due to a nonsense mutation Rho is responsible for silencing horizontally

transferred DNA elements, some of which aredetrimental to the host

Rho-Dependent Termination Sites


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Transcription Termination by Rho

E Nudler (2002) Genes Cells 7: 755

1. Rho monomers attach to unstructured nascenttranscripts

2. Using ATP, RNA wraps around the internal surface of

the Rho hexamer (or the outer rim)

3. Wrapping might pull the RNA-3 end from the RNAP

active centre or Rho may invade the active centre


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Damage-Dependent Termination

Mfd = Mutation frequency decline; 130 kDa

monomer

Mfd consists of three functional domains:

-DNA-binding domain

- ATPase domain

- RNAP-binding domain

Mfd recruits the DNA excision repair to the site

of DNA damage


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J. Roberts (2004) Curr. Opin. Microbiol. 7: 120

Effect of the DNA Translocase Activity

of Mfd on the RNA Polymerase


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V Pags (2003) DNA Repair 2: 273

Readthrough Transcription

Definition:A downstream gene will be transcribed at a

reduced rate due to a transcriptional terminator

5-10%


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6.2.5 Antitermination and

Attenuation of Transcription

AntiterminationThe default pathway results in premature

termination, and a regulatory moleculepromotes transcription readthrough

AttenuationThe default pathway is readthrough, and a

regulatory molecule induces transcription

termination


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1. Attenuation of the E. coli trp operon2. Attenuation of the B. subtilis trp operon

3. Antiterminator protein N of phage4. Antiterminator protein Q of phage

5. Antitermination of the bgl operon of E. coli

6. tRNA-mediated antitermination of Gram-

positive bacteria

Examples:

St t f th O d f


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Structure of the t r pOperon and of

Trp and Other Leader Peptides


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Features of the Regulatory Leader

Transcript of theE. c o l i t r p

Operon

Leader transcript: 141 nucleotides long

Can form three alternative RNA secondary

structures 1:2 = pause or antiterminator structure

2:3 = antiterminator structure

3:4 = terminator structure (intrinsic terminator)

14 amino acids long


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Transcription Attenuation of the E. co l i

t r pOperonPause allows a ribosome to

bind which disrupts thepausing RNAP complex


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Attenuation of the t r pOperon

Principle:

Formation of alternative secondary structures

Regulator:

Tryptophanyl-tRNA

M d l f T i ti Att ti f th


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Model of Transcription Attenuation of the

B. subtilis trp Operon

P. Gollnick (2002) BBA 1577: 240

TRAP: 11-mer

Binds 11 Trp


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Organization of the Phage Genome

Early transcription starts from two divergent

promoters PL and PR

Most of the two early transcripts are

terminated at strong terminators located after

the genes N and cro

St ct e of the Phage Antite


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Structure of the Phage Antiter-

mination Complex

Nus = N utilization substanceN binds to nut (N utilization): boxA and boxB


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The N Protein of Phage

N protein modifies RNAP into a termination-resistant from that allows readthrough of

downstream termination signals

N antiterminates at both Rho-dependent and

intrinsic termination sites


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Mechanisms of Antitermination

At Rho sites:

Antipausing speeds RNAP through the critical

release sites

By blocking some step of termination

At intrinsic terminators:

By directly preventing the formation of thehairpin

Structure of Phage Q Anti


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Structure of Phage Q Anti-

Termination Region


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Paused Complex With Q

Structure of Phage Q Antitermination


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Structure of Phage Q Antitermination

RegionTTGACTTATTGAATAAAATTGGGTAAATTTG

+1

ACTCAACGATGGGTTAATTCGCTCGTTGTG

pause site

GTAGTGAG ATG

1. Elongating RNAP pauses

2. Q binds to qut (in blue)

3. Q shifts RNAP into a new -35 region

(underlined) immediately upstream of the start

codon


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Model for Antitermination Control

by the BglG Antiterminator Protein

P. Gollnick (2002) BBA 1577: 240

Ribonucleic AntiTermination

BglG stabilizes an alternativestructure

M d l f A tit i ti C t l f


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Model for Antitermination Control of

b g lOperon Expression


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Conclusion

The antiterminator protein occurs in two forms

An active form (dimer)

An inactive form (phosphorylated monomer)

Active BglG stabilizes an alternative

secondary structure

Model of the Interaction of Uncharged


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Model of the Interaction of Uncharged

tRNAThr with the B . s u b t i l i s t h r S Leader

RNA

Regulation of Aminoacyl tRNA


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Regulation of Aminoacyl tRNA

Synthetase Operons in Gram-positive

Bacteria by Antitermination

Intrinsic terminatorstructures in the mRNA

leader sequences

Folding in alternative

structures

T B T i ti A tit i ti


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T Box Transcription Antitermination

~250 genes identified in Gram-positive

bacteria contain an about 300 n leader region

able to form a conserved secondary structure Most of these genes are involved in amino

acid biosynthesis Uncharged cognate tRNA interacts with the

leader region in at least two places to

establish an antiterminator structure

Specificity is achieved by binding of the

anticodon of the tRNA to a specifier codonbulged out of the secondary structure


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6.2.6 Regulators of Transcription

Three classes of transcriptional regulators:

1. Sigma factors

2. Transcriptional repressors

3. Transcriptional activators

T i ti l R l t Bi d t


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Transcriptional Regulators Bind to

DNA Using an Helix-Turn-Helix Motif

Gly

Most DNA-Binding Proteins Are Dimers


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Most DNA Binding Proteins Are Dimers

Classification of Known E co l i


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Classification of Known E. co l i

Transcription Factors

Of 71 transcription factors are

1. 18 activators

2. 20 repressors

3. 33 dual regulators

What determines whether a


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at dete es et e a

transcription factor acts as an

activator or a repressor ?

The position of the binding site relative to thetranscription start site

Activator binding sites:

Located in most cases upstream of the promoter

Repressor binding sites:

Located within or downstream of the promoter or

upstream in conjunction with a downstream site

E l i t i ti f t


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E. co l i transcription factors:

More than 300 transcription factors

Seven transcription factors (ArcA, CRP, Fis,

FNR, IHF, Lrp, NarL) control 50% of all

regulated genes

~60 transcription factors control only a single

promoter

Mechanisms of Repression


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Mechanisms of Repression

1. By steric hindrance

2. By a post-RNAP binding step:

- repression at the open complex

formation step

- repression at the promoter clearance

step3. By DNA looping

4. By modulation of an activator

Repression by Steric Hindrance


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Repression by Steric Hindrance

DF Browning (2004) Nature Rev. Microbiol. 2:1

Steric hindrance = Inhibiting RNAP binding tothe promoter

Repressing by DNA Looping


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Looping = Binding to distal sites and

interacting by looping

Repression by Modulation of an


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Activator

Modulation of an activator= Repressor binds to

an activator and prevents the activator from

functioning


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How is the activity of the repressor

modulated ?

i i l


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Transcriptional Repressors

1. The LacI repressor

2. The ArgR repressor

3. The RheA repressor

4. The HspR repressor5. The OmpR repressor

6. The LexA repressor7. The GalR repressor

The E. co l i l a c I Repressor


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p

LacI repressor = Tetramer


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Principle:

The inducer inactivates the

repressor

Inducers of the LacI Repressor


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p

Location of the Three l a cOperators


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Repression factor by binding to O1 only: 20-fold

Repression factor by binding to O1 and either O2

or O3: 50-fold

NA Becker (2005) J.

Mol.Biol. 349: 716

Absence of Lactose


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Absence of Lactose

CJ Wilson (2007) Cell. Mol. Life Sci. 64: 3

Presence of Lactose and Glucose


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Presence of Lactose and Glucose


Presence of Lactose


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Presence of Lactose


The E. co l i a r g R Repressor


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Principle:

The end product activates therepressor

The RheA Repressor


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At low temperature: The repressor prevents

expression of hsp18 and autoregulates its own

expression

At high temperature: The repressor is inactive


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Principle:

The repressor acts as thermosensor

The HspR Repressor


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The HspR Repressor

DnaK is Needed as a Corepressor


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P i i l


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Principle:

The corepressor is titrated bydenatured proteins

The OmpR


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The OmpR

Repressor

Target genes:

ompF

micF

Principle:


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p

The repressor becomes active after

phosphorylation

The LexA Repressor


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Principle:


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Principle:

The repressor cleaves itself after

interaction with activated RecA (RecA*)

protein = proteolytic cleavage

Pathway of Repressosome Formation


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Pathway of Repressosome Formation

S Roy (2005) Biochem. 44: 5373

Principle:


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Principle:

The repressor is inactivated by the

inducer galactose = conformational

change

6.2.6.2 Activation of Transcription

by Positive Regulators


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by Positive Regulators

(Transcriptional Activators)

Three different mechanisms:

1. Recruitment of RNAP

- activation at simple promoters

- activation at complex promoters2. Pre-recruitment of RNAP

3. Promoting isomerization of RNAP

Transcription Activation by


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Recruitment of the RNAP

Principle:

Promoter is weak causing no or insufficient

binding of RNAP

Activator binds first and then recruits the RNAP

Activation of Simple Promoters


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1. Class I activation

2. Class II activation

3. Activation by a conformational change

Example: CAP

Class I Activation


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The activator is bound to an upstream site and

contacts the CTD of the RNAP

Example: CAP

The CAP Activator


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Catabolite Activator Protein (CAP) = Cyclic AMP

Receptor Protein (CRP)

Functions as a homodimer in the presence of

the allosteric effector cAMP

CAP-binding site: 22 bp inverted repeat; each

half recognized by one monomer

Induces a bend in the DNA of about 90

Class II Activation


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The activator binds to a target site adjacent to thepromoter 35 hexamer interacting with domain 4

of 70Example: CI of phage

Activation by Conformation Change


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Activator binds at or very near the promoter

MerR-type activators twist the DNA to

reoriente the 35 and 10 elements

Activation at Complex Promoters


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1. Repositioning of an activator2. Co-dependence on independent contacts by two

activators3. Co-dependence due to co-operative binding of

activators

4. Co-dependence due to bacterial nucleoid proteins

5. Modulation by an epigenetic mechanism

6. Anti-activators

7. Subcellular relocalization of transcription factors

Repositioning of a Primary by a


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Secondary Activator

Activator 1 binds to a site where it is unable

to activate transcription

Activator 2 repositions activator 2

Example: MalT is repositioned by CAP

Co-dependence on Independent


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Contacts by Two Activators

Activator 1 is unable to contact the RNAP

Activator 2 bends the DNA

Co-dependence on Independent

Contacts by Two Activators


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Architecture at the E. coli proP P2 promoter

SM McLeod (2002) J. Mol. Biol. 316: 517

Co-Dependence Due to Co-

Operative Binding of Activators


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Activation of the E. coli melAB operon is

dependent on two different activators

TA Belyaeva (20002) Mol. Microbiol. 36: 211

- melibiose + melibiose

Co-Dependence Due to Bacterial

Nucleoid Proteins


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Activation of the E.

coli nir promoter

DF Browning (2000)

Mol. Microbiol. 37:

1258

A

B

odulation by an Epigenetic Mechanism


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A Barnard (2004) Curr. Opin. Microbiol. 7: 102

Anti-Activators


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Definition:

Protein which forms a complex with the

activator thereby preventing it from interacting

with the DNA

Examples:

NifA (activator) NifL (anti-activator)

PspF (activator) PspA (anti-activator)

The NifA - NifL Pair


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NifA: N-dependent activator of nitrogen fixationgenes

NifL: anti-activator; prevents transcription under

detrimental environmental conditions

J Barrett (2001) Mol. Microbiol. 39: 480

The PspF PspA Pair


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AJ Darwin (2005) Mol. Microbiol. 57: 621

PspB or/and PspC

may sense aninducing signal

Subcellular Relocalization of the

Activator


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A Bhm (2004) Curr. Opin. Microbiol. 7: 151

The Maltose System


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Consists of ten genes involved in uptake and

utilization of maltose and maltodextrins LamB acts as a specific diffusion pore

Uptake is mediated by a periplasmic-binding

protein-dependent ABC transporter

MalSQPZ: degrade maltose to glucose and

glucose -1-phosphate

The Regulatory Network Controlling

the Activity of MalT


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red MalT = inactive

green MalT = active

Transcription Activation by Pre-


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Recruitment

Definition:

1. RNAP holoenzyme and the transcriptional

activator form a complex in solution (in thecytoplasm)

2. This complex screens for the appropriatepromoter

The SoxS Activator of E. co l i


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Expression of soxS activated by oxidative stress

SoxS binds to a DNA sequence called Soxbox The Soxbox occurs more than 600 times on the

chromosomal DNA of E. coli Question: How does SoxS recognize the

appropriate Soxboxes ? Answer: By pre-recruitment

The Pre-Recruitment Mechanism


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Transcription Activation by

Isomerization of the RNA


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Polymerase

Components:

1. EN (54)2. Transcriptional activator

- with DNA-binding domain (binds to an

enhancer)

- without DNA-binding domain

The Isomerization Step Needs an

Activator


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S Wigneshweraraj (2008) Mol. Microbiol. 68: 538

E54 is unable to isomerize spontaneously

Most Activators Consist of Three

Functional Domains


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1. An N-terminal regulatory domain

2. A central ATPase (AAA+) which binds ATP

and a transcriptional activation domain

3. A C-terminal DNA-binding domain (HTH)

Some activators lack a DNA-binding domain

Enhancer-

D d


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DependentActivation

LL Beck (2007) Trends

Microbiol. 15: 530

Enhancer-

I d d t


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IndependentActivation

LL Beck (2007) Trends

Microbiol. 15: 530

Activator does not

contain a DNA-binding

domain

Function of the ATPase Domain


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The activator ATPase functions to remove a set

of repressive nucleoprotein interactions

Mechanisms controlling the

i t ti f th l t d i


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interaction of the regulatory domainwith the ATP-binding domain:

1. The regulator domain is phosphorylated by

a kinase (part of a two-component signal

transduction system)

Example one: NtrC (Nitrogen assimilation

regulator C)Phosphorylation causes oligomerization

Example two: DctD (Dicarboxylic acid

transport regulation D)


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2. The regulator binds a small ligandExample one: XylR of Pseudomonas putida

XylR binds toluene

Example two: NifA

NifA binds 2-oxoglutarate

NifA forms a repressive complex with NifL

transport regulation D)Phosphorylation relieves the inhibitory

effect of the N-terminal domain on the 54

interaction module

Interactions

Between NifL


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and NifA in

Response to

Environmental

Cues

Review Articles


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M Rappas (2007) Curr. Opin. Struct. Biol. 17: 110

LL Beck (2007) Trends Microbiol. 16: 530

S Wigneshweraraj (2008) Mol. Microbiol. 68: 538

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62 Gene Regulation