8.1 major modes of regulation gene expression: transcription of gene into mrna followed by...

8.1 Major Modes of Regulation

• Gene expression: transcription of gene into mRNA followed by translation of mRNA into protein (Figure 8.1)

• Most proteins are enzymes that carry out biochemical reactions

• Constitutive proteins are needed at the same level all the time

• Microbial genomes encode many proteins that are not needed all the time

• Regulation helps conserve energy and resources

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Figure 8.1 Upstreamregion

Downstreamregion

Start oftranscription Shine-Dalgarno

sequence (ribosome-binding site)

Start codon:Translationstarts here

Stop codon:Translation ends here

Transcriptionterminator

Transcription

Translation

Protein

DNA

mRNA

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8.2 DNA-Binding Proteins

• mRNA transcripts generally have a short half-life– Prevents the production of unneeded proteins

• Regulation of transcription typically requires proteins that can bind to DNA

• Small molecules influence the binding of regulatory proteins to DNA– Proteins actually regulate transcription

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8.2 DNA-Binding Proteins

• Most DNA-binding proteins interact with DNA in a sequence-specific manner

• Specificity provided by interactions between amino acid side chains and chemical groups on the bases and sugar–phosphate backbone of DNA

• Major groove of DNA is the main site of protein binding

• Inverted repeats frequently are binding site for regulatory proteins

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8.2 DNA-Binding Proteins

• Homodimeric proteins: proteins composed of two identical polypeptides

• Protein dimers interact with inverted repeats on DNA– Each of the polypeptides binds to one inverted

repeat (Figure 8.2)

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Figure 8.2

Domain containing protein–proteincontacts, holding protein dimer together

DNA-binding domain fits inmajor grooves and alongsugar–phosphate backbone

Inverted repeats

Inverted repeats

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8.2 DNA-Binding Proteins

• Several classes of protein domains are critical for proper binding of proteins to DNA

– Helix-turn-helix (Figure 8.3)• First helix is the recognition helix• Second helix is the stabilizing helix• Many different DNA-binding proteins from Bacteria

contain helix-turn-helix– lac and trp repressors of E. coli

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Figure 8.3

Stabilizinghelix

Recognitionhelix

Subunitsof bindingprotein

Turn

DNA

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8.2 DNA-Binding Proteins

• Multiple outcomes after DNA binding are possible1. DNA-binding protein may catalyze a specific

reaction on the DNA molecule (i.e., transcription by RNA polymerase)

2. The binding event can block transcription (negative regulation)

3. The binding event can activate transcription (positive regulation)

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8.3 Negative Control of Transcription: Repression and Induction

• Several mechanisms for controlling gene expression in bacteria– These systems are greatly influenced by

environment in which the organism is growing

– Presence or absence of specific small molecules

– Interactions between small molecules and DNA-binding proteins result in control of transcription or translation

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• Negative control: a regulatory mechanism that stops transcription– Repression: preventing the synthesis of an

enzyme in response to a signal (Figure 8.5)• Enzymes affected by repression make up a

small fraction of total proteins • Typically affects anabolic enzymes

(e.g., arginine biosynthesis)

8.3 Negative Control of Transcription: Repression and Induction

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8.3 Negative Control of Transcription: Repression and Induction

• Negative Control (cont’d)– Induction: production of an enzyme in response to

a signal (Figure 8.6)• Typically affects catabolic enzymes (e.g., lac

operon)• Enzymes are synthesized only when they are

needed– no wasted energy

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Figure 8.5

Repression

Cell numberTotal protein

Arginine added

Argininebiosynthesisenzymes

Time

Re

lati

ve

inc

rea

se

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Figure 8.6

Induction

Cell number

Total protein

Lactose added

-Galacto-sidase

Time

Re

lati

ve

inc

rea

se

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8.3 Negative Control of Transcription: Repression and Induction

• Inducer: substance that induces enzyme synthesis• Corepressor: substance that represses enzyme

synthesis• Effectors: collective term for inducers and

repressors• Effectors affect transcription indirectly by binding

to specific DNA-binding proteins– Repressor molecules bind to an allosteric

repressor protein– Allosteric repressor becomes active and binds to

region of DNA near promoter called the operator© 2012 Pearson Education, Inc.

Figure 8.7

RNApolymerase

Repressor

Repressor

RNApolymerase

Corepressor(arginine)

Transcription proceeds

Transcription blocked

arg Promoter arg Operator argC argB argH

arg Promoter arg Operator argC argB argH

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Figure 8.8

RNApolymerase

Repressor

Repressor

RNApolymerase

Inducer

Transcription proceeds

Transcription blocked

lac Promoter lac Operator lacZ lacY lacA

lac Promoter lac Operator lacZ lacY lacA

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8.4 Positive Control of Transcription

• Positive control: regulator protein activates the binding of RNA polymerase to DNA (Figure 8.9)

• Maltose catabolism in E. coli– Maltose activator protein cannot bind to DNA

unless it first binds maltose

• Activator proteins bind specifically to certain DNA sequence– Called activator-binding site, not operator

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Figure 8.9

Activator-binding site

RNApolymerase

Transcription proceeds

mal Promoter malE malF malG

No transcription

Activator-binding site

Maltose activator protein

Maltose activator protein

Inducer

RNApolymerase

mal Promoter malE malF malG

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8.4 Positive Control of Transcription

• Promoters of positively controlled operons only weakly bind RNA polymerase

• Activator protein helps RNA polymerase recognize promoter– May cause a change in DNA structure

– May interact directly with RNA polymerase

• Activator-binding site may be close to the promoter or several hundred base pairs away (Figure 8.11)

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Figure 8.11

Activator-binding site

Activatorprotein

Activator-binding site

Activator protein

RNApolymerase

RNApolymerase

Promoter

Promoter

Transcriptionproceeds

Transcriptionproceeds

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8.5 Global Control and the lac Operon

• Cyclic AMP and CRP– In catabolite repression, transcription is controlled

by an activator protein and is a form of positive control (Figure 8.14)

– Cyclic AMP receptor protein (CRP) is the activator protein

– Cyclic AMP is a key molecule in many metabolic control systems

• It is derived from a nucleic acid precursor• It is a regulatory nucleotide

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Figure 8.14 CRP protein

cAMP

RNApolymerase

lac Structural genes

Activerepressor

Inducer

Inactiverepressor

Lactose catabolism

DNA

mRNA mRNA

TranscriptionTranscription

TranslationTranslation

lacZ lacY lacAlacI

Activerepressorbinds tooperatorandblockstran-scription

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8.5 Global Control and the lac Operon

• Dozens of catabolic operons affected by catabolite repression– Enzymes for degrading lactose, maltose, and

other common carbon sources

• Flagellar genes are also controlled by catabolite repression– No need to swim in search of nutrients

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8.7 Two-Component Regulatory Systems

• Prokaryotes regulate cellular metabolism in response to environmental fluctuations– External signal is transmitted directly to the target

– External signal detected by sensor and transmitted to regulatory machinery (Signal transduction)

• Most signal transduction systems are two-component regulatory systems

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8.7 Two-Component Regulatory Systems

• Two-component regulatory systems (Figure 8.16)– Made up of two different proteins:

• Sensor kinase: (in cytoplasmic membrane) detects environmental signal and autophosphorylates

• Response regulator: (in cytoplasm) DNA-binding protein that regulates transcription

– Also has feedback loop• Terminates signal

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Figure 8.16Environmental signal

Phosphataseactivity

Transcription blockedRNApolymerase

Sensorkinase

Cytoplasmicmembrane

Response regulator

DNA

Promoter Operator Structural genes© 2012 Pearson Education, Inc.

8.7 Two-Component Regulatory Systems

• Almost 50 different two-component systems in E. coli– Examples include phosphate assimilation,

nitrogen metabolism, and osmotic pressure response

• Some signal transduction systems have multiple regulatory elements

• Some Archaea also have two-component regulatory systems

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8.9 Quorum Sensing

• Prokaryotes can respond to the presence of other cells of the same species

• Quorum sensing: mechanism by which bacteria assess their population density

– Ensures sufficient number of cells are present before initiating a response that requires a certain cell density to have an effect (e.g., toxin production in pathogenic bacterium)

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8.9 Quorum Sensing

• Each species of bacterium produces a specific autoinducer molecule (Figure 8.18)– Diffuses freely across the cell envelope

– Reaches high concentrations inside cell only if many cells are near

– Binds to specific activator protein and triggers transcription of specific genes

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Figure 8.18

Acyl homoserine lactone (AHL)

AHLActivator protein

Other cellsof the samespecies

Chromosome AHL synthase

AHL

Quorum-specificproteins

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8.9 Quorum Sensing

• Several different classes of autoinducers– Acyl homoserine lactone was the first

autoinducer to be identified

• Quorum sensing first discovered as mechanism regulating light production in bacteria including Aliivibrio fischeri (Figure 8.19)

– Lux operon encodes bioluminescence

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Figure 8.19

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8.9 Quorum Sensing

• Examples of quorum sensing– P. aeruginosa switches from free living to

growing as a biofilm

– Virulence factors of Staphylococcus aureus

• Quorum sensing is present in some microbial eukaryotes

• Quorum sensing likely exists in Archaea

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8.11 Other Global Control Networks

• Several other global control systems – Aerobic and anaerobic respiration

– Catabolite repression

– Nitrogen utilization

– Oxidative stress

– SOS response

– Heat shock response

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8.11 Other Global Control Networks

• Heat shock response:– Largely controlled by alternative sigma factors

(Figure 8.21)

– Heat shock proteins: counteract damage of denatured proteins and help cell recover from temperature stress

• Very ancient proteins

• Heat shock response also occurs in Archaea

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Figure 8.21

RpoHDnaK

Degradation ofRpoH by protease

RpoH

RpoH isreleased

Proteins unfold athigh temperature

DnaK bindsunfoldedproteins

RpoH is freeto transcribeheat shockgenes

Hig

h t

em

pe

ratu

reL

ow

te

mp

era

ture

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IV. Regulation of Development in Model Bacteria

• 8.12 Sporulation in Bacillus• 8.13 Caulobacter Differentiation

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8.12 Sporulation in Bacillus

• Regulation of development in model bacteria– Some prokaryotes display the basic principle of

differentiation

• Endospore formation in Bacillus (Figure 8.22)– Controlled by 4 sigma factors

– Forms inside mother cell

– Triggered by adverse external conditions (i.e., starvation or desiccation)

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