Regulation of cell activity

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• • Translational regulation mechanisms Translational regulation mechanisms

• • Bacterial genetics Bacterial genetics

Systems Microbiology

Wednesday Oct 11 - Ch 8 -Brock

Regulation of cell activity

• • Transcriptional regulation mechanisms Transcriptional regulation mechanisms

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Image removed due to copyright restrictions.

See Figure 8-1 in Madigan, Michael, and John Martinko. Brock Biology of Microorganisms.

11th ed. Upper Saddle River, NJ: Pearson Prentice Hall, 2006. ISBN: 0131443291.

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Regulation of prokaryotic transcription

1. Single-celled organisms with short doubling times must respond extremely rapidly to their environment.

2. Half-life of most mRNAs is short (on the order of a few minutes).

3. Coupled transcription and translation occur in a single cellular compartment.

Therefore, transcriptional initiation is usually the major control point.

Most prokaryotic genes are regulated in units called operons (Jacob and Monod, 1960)

Operon: a coordinated unit of gene expression consisting of one or more related genes and the operator and promoter sequences that regulate their transcription. The mRNAs thus produced are “polycistronic’—multiple genes on a single transcript.

DNA

Activator

Binding Site Repressor

Binding Site (Operator)

Promoter A B C

Regulatory Sequences Genes Transcribed as a Unit

Figure by MIT OCW.

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Initiation of transcription begins with promoter binding by RNAP holoenzyme

holoenzyme = RNAP core + Sigma

Diagram of RNA polymerase and transcription removed due to copyright restrictions.

Brock Biology of Microorganisms, vol. 11, Chapter 7

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Alternate Sigma Factors

recognize promoters of different architecture – different regulons of genes

Table and graphs removed due to copyright restrictions.

See Ishihama. Ann Rev Microbiol 54 (2000): 499-518.

Ishihama, 2000, Ann. Rev. Microbiol. 54:499-518

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Transcription factors interact with different components of RNAP in addition to sigma factor…..

Diagram removed due to copyright restrictions.

See Ishihama. Ann Rev Microbiol 54 (2000): 499-518.

Ishihama, 2000, Ann. Rev. Microbiol. 54:499-518

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Microbiology 151 (2005), 3147-3150; DOI 10.1099/mic.0.28339-0 Genome update: sigma factors in 240 bacterial genomes

Types of sigma factors…

Graphs removed due to copyright restrictions.

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Transcription factors

Typically DNA binding proteins that associate with the regulated promoter and either decrease or increase the efficiency of

transcription, repressors and activators, respectively - A

significant number of regulators do either one depending on conditions

TGTGTGGAATTGTGAGCGGATAACAATTTCACACA ACACACCTTAACACTCGCCTA

5' 3' 5'

3'

Inverted repeats on the DNA Domain containing protein-protein contacts,

holding protein dimer together

DNA binding domain fits in major grooves and along phosphate backbone

TGTTAAAGTGTGT T

Figure by MIT OCW.

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In presence of arginine, arginine biosynthesis genes are repressed

Time Repression

Relative Increase

Repression

No. cells

Arginine Biosynthesis

Total protein

Arginine added

Enzymes involved

in arginine synthesis Induction

Figure by MIT OCW.

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In the presence of lactose, lactose metabolizing genes are induced

Time

Relative Increase

Lactose Degradation Induction

No. cells

Total protein

Lactose added

β-Galactosidase

Figure by MIT OCW.

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The metabolism of lactose in E. coli & the lactose operon

•To use lactose as an energy source, cells must contain the enzyme β-galactosidase.

•Utilization of lactose also requires the enzyme lactose permease to transport lactose into the cell.

•Expression of these enzymes is rapidly induced

~1000-fold when cells are grown in lactose compared to glucose.

LacZ: β-galactosidase; Y: galactoside permease;

A: transacetylase (function unknown).

P: promoter; O: operator.

LacI: repressor; P_I and LacI are not part of the operon.

IPTG: non- metabolizable artificial inducer (can’t be cleaved)

H

H H

OH OH

HO OH

CH₂OH O

H

H H

OH OH

HO

CH₂OH O

H

H H

OH OH

HO

CH₂OH O

H

H H

OH OH

CH₂OH O

OH HO

H

H H

OH

H

H H

OH HO OH

CH₂OH

O OH OH

CH₂ O

Inside Outside

Lactose

Galactose Glucose

Allolactose b-galactosidase b-galactosidase

Trans-glycosylation

O

+

Lactose Galactoside permease

figure by MIT OCW.

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Negative regulation of the lac operon

Negative regulation: The product of the I gene, the repressor, blocks the expression of the Z, Y, and A genes by

interacting with the operator (O).

The inducer (lactose or IPTG) can bind to the repressor, which induces a conformational change in the repressor, thereby preventing its

interaction with the operator (O). When this happens, RNA polymerase is free to bind to the promoter (P) and initiates transcription of the lac genes.

How do we know that lacI encodes a trans-acting repressor?

RNA polymerase

lac operon

lac repressor DNA

Medium

Lactose

mRNA

Nascent polypeptide chains

Lactose permease

Thiogalactoside transacetylase

β-galactosidase P

I O Z Y A

Figure by MIT OCW.

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Symmetry matching between the tetramers of lac repressor and the nearly palindromic sequence of the lac operator

Each monomeric unit of lacI is 37-kD

Image of the X-ray structure of lac repressor tetramer bound to two 21-bp segments of DNA removed due to copyright restrictions.

The lac operator sequence is a nearly perfect inverted repeat centered around the GC base pair at position + 11.

5' ATGT TGTGTGGAATTGTGAGC GGATAACAATTTCACACAGGAA 3'

-10 +1 +10 +20

Half-site

+30

3' TACAACACACCTTAACACTCGC CTATTGTTAAAGTGTGT CCTT 5'

Figure by MIT OCW.

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“Diauxic” growth -

preferential use of one carbon source over another, in sequential fashion

Number of Viable Cells

Time of Incubation (hr) Glucose exhausted

Glucose and lactose

added Growth on glucose

Growth on lactose

Lactose exhausted

Figure by MIT OCW.

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Regulation of the lac operon involves more than a simple on/off switch provided by lacI/lacO

Observation: Glucose is a preferred sugar for E. coli, which uses glucose and ignores lactose in media containing both sugars. In these cells, β- galactosidase level is low, suggesting that derepression at the operator site is not enough to turn on the lac operon. This phenomenon is called catabolite repression.

This involves regulation of cyclic AMP (cAMP) levels, and its interaction with the Catabolite Activator Protein, CAP.

cAMP-CAP complex activates lac gene expression.

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Cooperative binding of cAMP-CAP and RNAP on the lac promoter

cAMP-CAP contacts the α-subunits of RNAP and enhances the binding of RNAP to the promoter.

cAMP-CAP

-84 -50 +1

a submit RNA polymerase

Lac control region

CAP site Polymerase site

Figure by MIT OCW.

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X-ray structure of CAP - cAMP bound to DNA

Image of the X-ray structure of the CAP-cAMP dimer in complexwith DNA removed due to copyright restrictions.

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Catabolite control of the lac operon

CAP sites are also present in other promoters. cAMP- CAP is a global catabolite gene activator.

(a) Under conditions of high glucose, a glucose breakdown product inhibits the enzyme adenylate cyclase, preventing the conversion of ATP into cAMP.

(b) As E. coli becomes starved for glucose, there is no breakdown product, and

therefore adenylate cyclase is active and cAMP is formed.

(c) When cAMP (a hunger signal) is present, it acts as an allosteric effector, complexing with the CAP dimer.

(d) The cAMP-CAP complex (not CAP alone) acts as an activator of lac operon transcription by binding to a region within the lac promoter. (CAP = catabolite

activator protein; cAMP = cyclic adenosine monophosphate)

High glucose

Low glucose

Inactive adenylate cyclase

ATP No cAMP

Active adenylate cyclase

cAMP ATP

P O

Z Y A

CAP

cAMP cAMP

cAMP

+ (A)

(B)

(D) (C)

Figure by MIT OCW.

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Positive and negative regulation of the lac operon

Promoter Operator

Inducer-repressor Lactose

No glucose present (cAMP high); lactose present Glucose present (cAMP low); lactose present Glucose present (cAMP low); no lactose

Abundant lac mRNA CAP

cAMP CAP

Repressor

Z Y A

Very little lac mRNA

B

C A

Figure by MIT OCW.

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Transcriptional termination can be an important target for regulation

Release of polymerase and RNA

3' 5'

3'

3' 5'

5'

5' 5'

3'

Termination site reached;

chain growth stops 5'

3'

Figure by MIT OCW.

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The tryptophan trp operon: two kinds of negative regulation

trp,O trpL trpE trpD trpC trpB trpA

Control sites

mRNA (low trp levels)

Leader mRNA (high trp levels)

or

Attenuator

Structural genes

Promoter

-35 +1

Operator Start of transcription

Inactive repressor

RNA polymerase

Tryptophan

Active repressor

Genes are ON Genes are OFF

mRNA

Trp-repressor complex activated for DNA binding

Tryptophan + trp repressor dimer

Binds Operator; blocks RNAP binding &

represses transcription;

Tryptophan a co-repressor

Figure by MIT OCW.

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Image of trp and DNA removed due to copyright restrictions.

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Many genes terminate transcription at sequences downstream of the coding sequence

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Attenuation and the Tryptophan Operon

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Attenuation is mediated by the tight coupling of transcription and translation

•The ribosome translating the trp leader mRNA follows closely behind the RNA polymerase that is transcribing the DNA template.

•Alternative conformation adopted by the leader mRNA.

•The stalled

ribosome is waiting for tryptophanyl- tRNA.

•The 2:3 pair is not an attenuator and is more stable than the 3:4 pair.

High tryptophan

Leader peptide completed

Transcription terminator

UUU 3, trpL mRNA Ribosome transcribing

the leader peptide mRNA and blocking sequence 2

"Terminated"

RNA polymerase +

Low tryptophan

Antiterminator

trp operon mRNA

Ribosome stalled at tandem Trp codons Incomplete

leader peptide Transcribing RNA polymerase

DNA encoding trp operon

1

1 2

2

3

4

Figure by MIT OCW.

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The arabinose (ara) operon

• 3 genes encoding enzymes of the arabinose degradation pathway

arabinose – (1) araA => L-arabinose isomerase

– (2) araB => L-ribulose kinase

– (3) araD => L-ribulose 5-phosphate

• Regulatory elements

– araO1, araO2

– araI (I for inducer) – P_BAD promoter

arabinose

L-ribulose

L-ribulose –5-P

D-xylulose –5-P

Pentose P Pathway

3 1

2

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The arabinose (ara) operon of Escherichia coli

P P

t t

CRP

O₂ O₁ I₁ I₂

araC araB araA araD

AraC dimer

Activator/

Repressor

Interaction of AraC with O and I sites determines transcription at the Ara locus

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Key features of Arabinose regulation 1. Arabinose is a positive regulator of transcription.

2. In the absence of arabinose AraC binds regulatory sites I₁and O_2.

This represses AraBAD operon transcription (DNA looping).

3. AraC levels are autoregulated by excess AraC binding to O₁ 4. When arabinose is bound to AraC, AraC binds I₁ and I₂.

5. When Glucose is absent, cAMP levels rise and cAMP-CRP bind adjacent to I1.

Together these events trigger AraBAD expression.

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ara Transcriptional control-1

P P

t O₂ O₁ CRP t

I₁ I₂

araC araB araA araD

1. No L-arabinose

O₂

I₁ I₂ O₁

repressed repressed P ^araC P ^araBAD

AraC dimer is flexible

O₂

I₁ I₂ O₁

repressed Active

P ^araBAD P ^araC

AraC binds to O₂ and I₁ repressing P araBAD Excess AraC binds to O₁ regulating cellular levels of AraC protein

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ara Transcriptional control-2

P P

t O₂ O₁ CRP t

araC I₁ I₂ _araB _araA _araD

2. + Inducer L-arabinose + cAMP-CRP

P P ^araBAD ^induced (activated) t O₂ O₁

I₁ I₂

AraC binds to I₂and I₁ activating P araBAD

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Differences between the arabinose and lactose operons

1. AraC can act as both a repressor and activator of AraBAD expression.

This is an example of positive control.

2. AraC can regulate its own synthesis by repressing its own transcription.

This is a common feature of many genes.

3. Ara operon provides an example of regulation at a distance by DNA looping.

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Common mechanisms of regulation of transcription, with variations on a theme…

lac ara trp

Regulation ^negative_positive positive, auto-^negative, negative attenuation

Operators lacO1, O2, O3 araO1, O2, I trpO

CRP-site ⁺ ⁺ ^-

Regulator (location)

lacI upstream

araC

upstream trp R distant

Effector allolactose (activator)

arabinose (activator)

tryptophan (co-repressor)

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“Flipping promoters” Flagellar phase variation

Flagellar

Phase variation

Figure by MIT OCW.

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Environmentally-responsive adaptation

External stimuli

Internal stimuli Membrane- permeable signals

Transmembrane receptor

Indirect effect

Physiological change

Gene

Regulation

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Simple paradigm for environmental signalling – the two- component system

> 30 such systems in E. coli – also found in plants and fungi

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Basic model for a two component-regulatory system

Sensor histidine kinase (HK) – may or may not be transmembrane – phosphorylates itself

Response regulator (RR) – often, but not always affects gene expression – phosphorylated by HK

Hoch and Varughese, 2001, J. Bacteriol. 183:4941-4949

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The PhoR/PhoB two-component regulatory system in E. coli

In response to low phosphate

concentrations in the environment and periplasmic space, a phosphate ion dissociates from the periplasmic domain of the sensor protein PhoR.

This causes a conformational change that activates a protein kinase

transmitter domain in the cytosolic region of PhoR. The activated

Diagram removed due to copyright restrictions. transmitter domain transfers an ATP γ-phosphate to a histidine in the

transmitter domain. This phosphate is then transferred to an aspartic acid in the response regulator PhoB.

Phosphorylated PhoB then activate transcription from genes encoding proteins that help the cell to respond to low phosphate, including phoA, phoS, phoE, and ugpB.

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Two-components alone aren’t always sufficient –

phospho relays are through multiple protein modulators are a common regulatory mechanism

Stock et al. 2000, Ann. Rev. Genet 69:183-215

‘phosphorylation cascades’

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Allow response to wide range of chemical and

physical stimuli

Many variations on the basic theme exist and the more they are studied the more

permutations are observed

Stock et al. 2000, Ann. Rev. Genet 69:183-215

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CheP CheY

CheW CheW +CH₂

-CH₂

ATP CheA

CheZ CheB

Cell wall

Cytoplasmic membrane

Flagellar motor Transducer (MCP)

CheB P CheY P

CheA P

Attractants

Figure by MIT OCW.

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Quorum Sensing

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Substrates, Products and Pathways Involved in the Bacterial Bioluminescence Reaction hν

NAD(P)⁺ FMNH₂

RCHO + FMNH₂+ O₂

O₂

RCOOH + FMN + H₂O+ hν RCOOH

NADPH + H⁺

RCHO

ATP

AMP⁺ PPi NADP⁺ Luciferase

NAD(P)H + H⁺

Oxidoreductase

FMN

Luciferase Fatty Acid Reductase

Figure by MIT OCW.

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10⁸ 0 10⁹ Luminescence

( (

Quanta/sec OD 660

10¹⁰ 10¹¹

.2 .4

OD₆₆₀

.6 .8 1.0

10¹² 10¹³

MJ-1 pJE202 pJE201 pJE205 Growth

Figure by MIT OCW.

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Nucleic Acids Res. 1987 December 23; 15(24): 10455–10467.

Nucleotide sequence of the regulatory locus controlling expression of bacterial genes for bioluminescence

Negative Feedback

Operon L Operon R

lux R lux I lux CDABE

Positive Feedback

Regulatory Proteins

Model for Regulation of Bioluminescence

Enzymes for Bioluminescence

Figure by MIT OCW.

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Vibrio fischeri and luminescence :

LuxI

LuxR

LuxR Target Genes

Figure by MIT OCW.

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Quorum Sensing

R C CH

₂

C N H

Acyl Homoserine Lactone (AHL) O

H

O

H

O

Figure by MIT OCW.

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See Figure 8-22b in Madigan, Michael, and John Martinko. Brock Biology of Microorganisms.

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Examples of bacteria that use acylated homoserine lactones

Bacteria Function

Vibrio fischeri luminescence Aeromonas hydrophila proteases Agrobacterium tumefaciens conjugation Burkholderia cepacia siderophores Chromobacterium violaceum antibiotics Erwinia chrysanthemi pectinase Pseudomonas aereofaciens phenazines Pseudomonas aeruginosa biofilms, etc

Rhizobium etli number of nodules

Yersinia pseudotuberculosis agreggation and motility

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Variations and Complications : Vibrio harveyi

LuxLM LuxS

LuxR LuxN

LuxO

X luxCDABE

LuxU

LuxQ LuxP

H D P

D

P

H D

P H P

P

Figure by MIT OCW.

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The “stringent response”

A type of translational global control

When bacteria are starved of nutrients, they immediately shut down gene expression and other metabolic activities.

1. Total RNA synthesis is reduced to ~ 10% of normal levels.

2. There is a massive >10-fold reduction in rRNA and tRNA transcription.

3. Protein synthesis decreases.

(The unusual nucleotides ppGpp and pppGpp accumulate during the stringent response).

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Stringent response in E. coli

1. Binding of an uncharged tRNA to the A-site

2. Binding of RelA to the 30S subunit

3. Synthesis of ppGpp

4. Downregulation / inhibition of transcription

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O

HO P OH

O

O P OH

O

O P O

OH

HO P O

O O

P OH

OH N

N NH N NH₂

OH O CH₂O O

The ppGpp inhibits the elongation phase of transcription.

The stringent factor RelA is a pppGpp synthetase that is associated with ~ 5% of ribosomes.

RelA protein produces one pppGpp every time the A site is occupied by an uncharged tRNA.

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Ribosomal protein L11 undergoes a conformational change when an uncharged tRNA binds.

This activates RelA stringent factor.

The unusual nucleotides ppGpp and pppGpp accumulate during the stringent response.

Total RNA synthesis is reduced to ~ 10% of normal levels.

There is a massive >10-fold reduction in rRNA and tRNA transcription.

Protein synthesis decreases.

(The SpoT protein degrades ppGpp so that normal gene expression can resume rapidly when conditions improve.)

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Review of gene regulation in bacteria.

With genes that are expressed constitutively,

promoter strength determines the level of expression.

DNA-binding proteins can switch genes on and off.

Repressors switch genes off.

[They prevent RNA pol from gaining access to promoters].

Activators switch genes on.

[They enable RNA pol to bind to promoters]

Activity of repressors and activators can be influenced by small molecules, temperature, phosphorylation etc.

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RNA secondary structure can control gene expression.

With attenuation, AA-tRNA availability influences early termination.

With riboswitches, small molecules influence early termination or translation intiation.

Alternative sigma factors bring about global changes in gene expression.

The stringent response shuts down gene expression when times are tough.

Promoter inversion can affect gene expression in pathogenic bacteria.