Gene Regulation in Gene Regulation in ProkaryotesProkaryotes

Outline of Chapter 16Outline of Chapter 16 There are many steps in gene expression and regulation There are many steps in gene expression and regulation

can occur at any one of themcan occur at any one of them Genetic and molecular studies show that most regulation Genetic and molecular studies show that most regulation

affects the initiation of RNA transcriptsaffects the initiation of RNA transcripts Studies of genes for lactose utilizationStudies of genes for lactose utilization

Negative regulation – blocks transcriptionNegative regulation – blocks transcription Positive regulation – increases transcriptionPositive regulation – increases transcription DNA binding proteins acting on RNA polymerase at promoter are main DNA binding proteins acting on RNA polymerase at promoter are main

agents of regulationagents of regulation

Attenuation of expression – tryptophan pathwayAttenuation of expression – tryptophan pathway Gene expression is fine tuned by premature termination of Gene expression is fine tuned by premature termination of

transcriptiontranscription

Outline of Chapter 16Outline of Chapter 16 Global regulatory mechanisms: Global regulatory mechanisms: E.E. coli’scoli’s

response to heat shock is an example of the response to heat shock is an example of the bacterial ability to coordinate the expression of bacterial ability to coordinate the expression of different sets of genes dispersed around the different sets of genes dispersed around the chromosome.chromosome. Microarray analysis is an important new tool for Microarray analysis is an important new tool for

detecting changes in gene expression in response to detecting changes in gene expression in response to environmental changesenvironmental changes

Comprehensive example: How Comprehensive example: How Vibrio choleraeVibrio cholerae regulate their virulence genesregulate their virulence genes

RNA polymerase is the key enzyme RNA polymerase is the key enzyme for transcriptionfor transcription

RNA polymerase involved in three phases of RNA polymerase involved in three phases of transcriptiontranscription Initiation – sigma subunit + core enzyme (two alpha, one beta, Initiation – sigma subunit + core enzyme (two alpha, one beta,

and one beta’ subunit)and one beta’ subunit) Binds to promoter, unwinds DNA, begins polymerization of bases Binds to promoter, unwinds DNA, begins polymerization of bases

complementary to DNA templatecomplementary to DNA template Elongation – movement away from promoter sigma subunit Elongation – movement away from promoter sigma subunit

released, polymerizationreleased, polymerization Termination –signal reached by RNA polymeraseTermination –signal reached by RNA polymerase

Rho dependent termination – Rho factor recognizes sequence in Rho dependent termination – Rho factor recognizes sequence in mRNA, binds to it, and pulls it away from RNA polymerasemRNA, binds to it, and pulls it away from RNA polymerase

Rho independent termination – stem loop structure formed by sequence Rho independent termination – stem loop structure formed by sequence of 20 bases with a run of 6 or more U’s signals release of RNA of 20 bases with a run of 6 or more U’s signals release of RNA polymerasepolymerase

Fig. 16.2

Translation in prokaryotes starts before transcription Translation in prokaryotes starts before transcription endsends Initiation sites for translation signal ribosomes to bind near 5’ Initiation sites for translation signal ribosomes to bind near 5’

end of mRNA while downstream transcription is still occurringend of mRNA while downstream transcription is still occurring Polycistronic mRNAs often lead to the translation of several Polycistronic mRNAs often lead to the translation of several

genes at the same time from one mRNA transcriptgenes at the same time from one mRNA transcript The regulation of gene expression can occur at many The regulation of gene expression can occur at many

stepssteps Binding of RNA polymerase to promoterBinding of RNA polymerase to promoter Shift from initiation to elongationShift from initiation to elongation Release of mRNA at terminationRelease of mRNA at termination Posttranscriptional stability of mRNAPosttranscriptional stability of mRNA Efficiency of ribosomes to recognize translation initiation sitesEfficiency of ribosomes to recognize translation initiation sites Stability of polypeptide productStability of polypeptide product

The utilization of lactose by The utilization of lactose by E. coliE. coli: : A model system for gene regulationA model system for gene regulation

The presence of lactose induces expression of the genes The presence of lactose induces expression of the genes required for lactose utilizationrequired for lactose utilization InductionInduction – stimulation of protein synthesis – stimulation of protein synthesis InducerInducer – molecule that stimulates synthesis – molecule that stimulates synthesis LactoseLactose – inducer of genes for lactose utilization – inducer of genes for lactose utilization

1950s and 1960s – Golden era of bacterial genetics1950s and 1960s – Golden era of bacterial genetics Advantages of Advantages of E. coliE. coli and lactose utilization system and lactose utilization system

Culture large numbers of bacteria allow isolation of rare mutantsCulture large numbers of bacteria allow isolation of rare mutants Lactose genes not essential for survival (can use glucose as carbon Lactose genes not essential for survival (can use glucose as carbon

source)source) Induction increases expression 1000 fold making mutant identification Induction increases expression 1000 fold making mutant identification

easyeasy Color changes using Color changes using -galactosidase enzyme (e.g., OPNG, X-gal) make -galactosidase enzyme (e.g., OPNG, X-gal) make

measurement of expression levels efficientmeasurement of expression levels efficient

Coordinate repression and induction of three genes Coordinate repression and induction of three genes revealed by studies of lactose-utilization mutantsrevealed by studies of lactose-utilization mutants

Jacques Monod and Francois Jacob – Jacques Monod and Francois Jacob – Pasteur Institute in ParisPasteur Institute in Paris Proposed Proposed Operon TheoryOperon Theory of gene regulation of gene regulation

Single signal can simultaneously regulate expression Single signal can simultaneously regulate expression of several genes that are clustered together on a of several genes that are clustered together on a chromosome and involve the same processchromosome and involve the same process

Because genes are clustered, they are transcribed Because genes are clustered, they are transcribed together as single mRNAtogether as single mRNA

Clusters of genes are called OperonsClusters of genes are called Operons

Complementation Analysis of mutants identifies Complementation Analysis of mutants identifies lactose utilization geneslactose utilization genes

Monod et al. isolated many LacMonod et al. isolated many Lac-- mutants unable to mutants unable to utilize lactoseutilize lactose

Complementation analysis identified three genes Complementation analysis identified three genes (lacZ, lacY, and lacA) in a tightly linked cluster(lacZ, lacY, and lacA) in a tightly linked cluster

Fig. 16.5

Experimental evidence for repressor proteinExperimental evidence for repressor protein

Isolated mutant in Isolated mutant in lacIlacI gene gene ConstitutiveConstitutive mutant – synthesized mutant – synthesized --

galactosidase and lac permease even in absence galactosidase and lac permease even in absence of lactose (inducer)of lactose (inducer)

lacIlacI must be a must be a repressorrepressor – cells must need – cells must need lacIlacI protein product to prevent expression of protein product to prevent expression of lacYlacY and and lacZlacZ in absence of inducer in absence of inducer

PaJaMo experimentPaJaMo experiment lacIlacI+, +, lacZlacZ+ x + x lacIlacI-- lacZlacZ-- F F--

Initial synthesis of Initial synthesis of --galactosidase stopsgalactosidase stops

Addition of inducer resumes Addition of inducer resumes synthesissynthesis

Conclusion – initial lack of Conclusion – initial lack of repressor allows synthesis. repressor allows synthesis. As As lacIlacI is transferred, is transferred, synthesis stopssynthesis stops

Repressor stops Repressor stops transcription by binding to transcription by binding to operatoroperator site near promoter site near promoter

Fig. 16.6

Inducer releases repressor to trigger Inducer releases repressor to trigger enzyme synthesisenzyme synthesis

Addition of lactose inducer caused Addition of lactose inducer caused – – galactosidase synthesis to continuegalactosidase synthesis to continue

Conclusion: Inducer binds to repressor so Conclusion: Inducer binds to repressor so repressor can not bind to DNArepressor can not bind to DNA

AllostericAllosteric effect - inducer bound to effect - inducer bound to promotor changes conformation of protein promotor changes conformation of protein so it can not bind to DNAso it can not bind to DNA

Repressor has binding domains for Repressor has binding domains for operator and for the induceroperator and for the inducer

Fig. 16.7

Changes in the operator can also Changes in the operator can also affect repressor activityaffect repressor activity

Fig. 16.8

Proteins act in Proteins act in transtransDNA sites act only in DNA sites act only in ciscis

TransTrans acting elementsacting elements can diffuse through can diffuse through cytoplasm and act at target DNA sites on cytoplasm and act at target DNA sites on any DNA molecule in cellany DNA molecule in cell

CisCis acting elementsacting elements can only influence can only influence expression of adjacent genes on same DNA expression of adjacent genes on same DNA moleculemolecule

Three experiments elucidate Three experiments elucidate ciscis and and transtrans

acting elements using F’ plasmidacting elements using F’ plasmid

Inducible synthesisInducible synthesis lacIlacI++ gene encodes a gene encodes a

diffusible element that diffusible element that acts in acts in transtrans by by binding to any binding to any operator it encounters operator it encounters regardless of regardless of chromosomal locationchromosomal location

Insert Figure 16.9ahere

Fig. 16.9 a

NoninducibleNoninducible

All operator sites (O+) All operator sites (O+) eventually occupied by eventually occupied by superrepressorsuperrepressor

lacIlacI supperrepressor can supperrepressor can not bind inducernot bind inducer

lacIlacIs s mutant encodes a mutant encodes a diffusible element that diffusible element that binds to operator binds to operator regardless of chromosomal regardless of chromosomal location (trans acting location (trans acting element)element)

Insert Figure 16.9bhere

Fig. 16.9 b

ConstitutiveConstitutive

Presence of OPresence of O++ plasmid plasmid does not compensate does not compensate for Ofor Occ mutation on mutation on bacterial chromosomebacterial chromosome

Operator is Operator is ciscis acting acting elementelement

Insert Figure 16.9chere

Fig. 16.9 c

The Operon TheoryThe Operon Theory

The playersThe players laczlacz, , lacYlacY, , lacZlacZ genes that split lactose into glucose and galactose genes that split lactose into glucose and galactose Promotor site to which RNA polymerase bindsPromotor site to which RNA polymerase binds cis acting operator sitecis acting operator site trans-acting repressor that can bind to operator (encoded by trans-acting repressor that can bind to operator (encoded by lacIlacI gene) gene) Inducer that prevents repressor from binding to operatorInducer that prevents repressor from binding to operator

Fig. 16.10 a

The Operon TheoryThe Operon Theory

RepressionRepression In absence of lactose, repressor binds to operator which In absence of lactose, repressor binds to operator which

prevents transcriptionprevents transcription Negative regulatory elementNegative regulatory element

Fig. 16.10 b

The Operon TheoryThe Operon Theory InductionInduction

Lactose presentLactose present Allolactose binds to Allolactose binds to

repressor.repressor. Repressor changes Repressor changes

shape and can not bind shape and can not bind to operatorto operator

RNA polymerase binds RNA polymerase binds to promotor and to promotor and initiates transcription of initiates transcription of polycistronic mRNApolycistronic mRNA

Fig. 16.10 c

Positive control increases Positive control increases transcription of transcription of lacZlacZ, , lacYlacY, and , and lacAlacA

cAMP binds to cAMP binds to CRP (cAMP CRP (cAMP receptor protein) receptor protein) when glucose is lowwhen glucose is low

CRP binds to CRP binds to regulatory regionregulatory region

Enhances activity Enhances activity of RNA polymerase of RNA polymerase at lac promotorat lac promotor

Fig. 16.11

Some positive regulators increase Some positive regulators increase transcription of genes in only one pathwaytranscription of genes in only one pathway

AraCAraC is a positive is a positive regulator for all regulator for all arabinose genes arabinose genes which break down which break down sugar arabinosesugar arabinose

Loss of function Loss of function mutation results in mutation results in little or no little or no expression of genesexpression of genes

Fig. 16.12

Molecular studies help fill in details Molecular studies help fill in details of control mechanismsof control mechanisms

Radioactive tag attached Radioactive tag attached to lac repressorto lac repressor Repressor from Repressor from lacIlacI++ cells cells

purified and mixed with purified and mixed with operator DNA, cosediment operator DNA, cosediment occurredoccurred

Repressor from Repressor from lacIlacI++ mixed mixed with mutant operator DNA, with mutant operator DNA, no cosediment occurredno cosediment occurred

Fig. 16.13

Many DNA-Binding proteins contain Many DNA-Binding proteins contain a helix-turn-helix motifa helix-turn-helix motif

Two Two αα-helical regions -helical regions separated by a turn in separated by a turn in the protein structurethe protein structure

Helix-turn-helix motif Helix-turn-helix motif fits into major groove fits into major groove of DNAof DNA

Most repressor Most repressor proteinsproteins

Fig. 16.14 a

Specific amino acids in the a-helix determine Specific amino acids in the a-helix determine the binding specificity of repressor proteinsthe binding specificity of repressor proteins

Hybrid 434-P22 repressor engineered to have amino acid Hybrid 434-P22 repressor engineered to have amino acid sequence that will bind to bacterial virus 434 and sequence that will bind to bacterial virus 434 and bacteriophage P22bacteriophage P22

Fig. 16.14 b

Most regulatory proteins are Most regulatory proteins are oligomericoligomeric

More than one binding domain

DNase footprint identifies binding region

DNase cannot digest protein covered sites

Fig. 16.15 a

The looping of DNA is a common The looping of DNA is a common feature of regulatory proteinsfeature of regulatory proteins

AraCAraC acts as both a acts as both a repressor and activatorrepressor and activator No arabinoseNo arabinose

Binding to Binding to araOaraO and and araIaraI11 causes looping and causes looping and

prevents RNA from prevents RNA from transcribingtranscribing

Arabinose presentArabinose present AraCAraC binds to binds to araIaraI11 and and

araIaraI22 bot not to bot not to araOaraO. RNA . RNA polymerase interacts with polymerase interacts with araCaraC at the at the araIaraI sites and sites and transcribes genestranscribes genes

Fig. 16.16

How regulatory proteins interact How regulatory proteins interact with RNA polymerasewith RNA polymerase

Negative regulators (Negative regulators (laclac repressor) repressor) Physically block DNA-binding sites of RNA Physically block DNA-binding sites of RNA

polymerasepolymerase Positive regulatorsPositive regulators Establish physical contact with RNA Establish physical contact with RNA

polymerase enhancing enzyme’s ability to polymerase enhancing enzyme’s ability to initiate transcriptioninitiate transcription

Using the lacZ gene as a reporter of Using the lacZ gene as a reporter of gene expressiongene expression

Reporter gene – protein encoding gene Reporter gene – protein encoding gene whose expression in the cell is quantifiable whose expression in the cell is quantifiable by techniques of protein detection.by techniques of protein detection.

Fusion of reporter gene to cis acting Fusion of reporter gene to cis acting regulatory regions allows assessment gene regulatory regions allows assessment gene activity by monitoring amount of reporter activity by monitoring amount of reporter gene productgene product

Fusion used to perform genetic studies of the Fusion used to perform genetic studies of the regulatory region of gene Xregulatory region of gene X

Fig. 16.18 a

Creating a Creating a collection of collection of

lacZ lacZ insertions in insertions in

the the chromosomechromosome

Fig. 16.18 b

Use of a fusion to Use of a fusion to overproduce a gene overproduce a gene

productproduct

Fig. 16.18 c

The attenuation of gene expression: Fine tuning of the The attenuation of gene expression: Fine tuning of the trptrp operon through termination of transcriptionoperon through termination of transcription

The presence of tryptophan activates a The presence of tryptophan activates a repressor of the trp operonrepressor of the trp operon trpR gene produces repressortrpR gene produces repressor

CorepressorCorepressor – tryptophan binds to trp repressor – tryptophan binds to trp repressor allowing it to bind to operator DNA and inhibit allowing it to bind to operator DNA and inhibit transcriptiontranscription

Termination of transcription fine Termination of transcription fine tunes regulation of tunes regulation of trptrp operon operon

trpRtrpR-- mutants are not constitutive mutants are not constitutive Repressor independent change in Repressor independent change in trptrp expression expression

Two alternative transcripts lead to different Two alternative transcripts lead to different transcriptional outcomestranscriptional outcomes Leader sequence can fold in two different stable Leader sequence can fold in two different stable

conformationsconformations Tryptophan present – ribosome moves quickly past codons in Tryptophan present – ribosome moves quickly past codons in

leader allowing stem-loop to form terminating transcriptionleader allowing stem-loop to form terminating transcription Tryptophan absent – ribosome stalls allowing normal stem Tryptophan absent – ribosome stalls allowing normal stem

loop structure to form and transcription proceeds normallyloop structure to form and transcription proceeds normally

Global regulatory mechanisms coordinate the Global regulatory mechanisms coordinate the expression of many genesexpression of many genes

Normal sigma factor (Normal sigma factor (7070) binds to RNA polymerase and ) binds to RNA polymerase and recognizes sequence in promoter to initiate transcriptionrecognizes sequence in promoter to initiate transcription

Heat shock disables Heat shock disables 7070

Product of Product of rpoHrpoH gene, gene, 3232 binds to sequence in promoter of binds to sequence in promoter of heat shock genes when heat stressed and starts transcriptionheat shock genes when heat stressed and starts transcription

Fig. 16.21 a

Factors influencing increase in Factors influencing increase in 3232 activity after heat shockactivity after heat shock

Increase in transcription of the rpoH geneIncrease in transcription of the rpoH gene Increase in the translation of Increase in the translation of 3232 mRNA mRNA

stemming from greater stability of rpoH mRNAstemming from greater stability of rpoH mRNA Increase in the stability and activity of the Increase in the stability and activity of the 3232

protein. Chaperones DnaJ/K bind and inhibit protein. Chaperones DnaJ/K bind and inhibit 3232 under normal conditions. At high temperature, under normal conditions. At high temperature, binding to binding to 3232 does not occur and more does not occur and more 3232 is free is free to associate with RNA polymerase.to associate with RNA polymerase.

Inactivity of Inactivity of 70 70 decreases competition with decreases competition with 3232 to to form RNA polymerase holoenzymeform RNA polymerase holoenzyme

What enables transcription of What enables transcription of 3232 during heat shock?during heat shock?

Normal temperatures, Normal temperatures, rpoHrpoH gene (encodes gene (encodes 3232) has promoter sequence recognized by ) has promoter sequence recognized by 7070 which starts transcriptionwhich starts transcription

High temperatures (no High temperatures (no 7070) a different promoter ) a different promoter sequence of the sequence of the rpoHrpoH gene is recognized by a gene is recognized by a different sigma factor, different sigma factor, 2424

SummarySummary

E. coli’sE. coli’s heat shock response is controlled by heat shock response is controlled by alternative sigma factors that recognize different alternative sigma factors that recognize different promoter sequencespromoter sequences

Alternative sigma factors bind to RNA polymerase Alternative sigma factors bind to RNA polymerase as temperatures change to start transcription of as temperatures change to start transcription of heat shock proteinsheat shock proteins

The induction of alternative sigma factors that The induction of alternative sigma factors that recognize different promoter sequences serve as recognize different promoter sequences serve as global control regulatory mechanisms in global control regulatory mechanisms in E. coliE. coli and many other bacteriaand many other bacteria

Microarrays – a tool for uncovering Microarrays – a tool for uncovering changes in gene expressionchanges in gene expression

Cellular responses to global environmental Cellular responses to global environmental changes can be measured by microarray changes can be measured by microarray analysis of mRNA isolated from cultures analysis of mRNA isolated from cultures grown in different environmental conditionsgrown in different environmental conditions

Comparisons of wild-type cultures with Comparisons of wild-type cultures with strains containing mutations in key strains containing mutations in key regulatory regions help identify genes and regulatory regions help identify genes and regulatory elements involved in response to regulatory elements involved in response to specific environmental changesspecific environmental changes

Fig. 1.13

Regulation of Virulence Genes in Regulation of Virulence Genes in V. choleraeV. cholerae

Bacterial agents of cholera sense changes in Bacterial agents of cholera sense changes in environment and transmit signals to environment and transmit signals to regulators that initiate, enhance, diminish, regulators that initiate, enhance, diminish, or repress expression of various genes.or repress expression of various genes.

Three regulatory proteins – ToxR, ToxS, Three regulatory proteins – ToxR, ToxS, and ToxT – turn on the genes for virulenceand ToxT – turn on the genes for virulence

Experiments generate model for regulation of Experiments generate model for regulation of virulence genes in virulence genes in V. choleraeV. cholerae

Cloned two genes encoding subunits of Cloned two genes encoding subunits of cholera toxin: cholera toxin: ctxActxA and and ctxBctxB

Made Made ctxA-lacZctxA-lacZ reporter gene fusion reporter gene fusion Created vector library of Created vector library of V. choleraeV. cholerae

genomic DNAgenomic DNA Used Used E. coliE. coli to perform genetic to perform genetic

manipulationsmanipulations

Isolated a gene that regulates expression of Isolated a gene that regulates expression of ctxctx operon operon Transformed Transformed E. coliE. coli containing containing ctx-lacZctx-lacZ construct with clones construct with clones

containing containing V. choleraeV. cholerae DNA DNA Clones that contain a positive regulator should turn on Clones that contain a positive regulator should turn on ctx-lacZctx-lacZ

constructconstruct Identified Identified ToxRToxR, a transmembrane protein, a transmembrane protein Identified Identified ToxSToxS, helps , helps ToxRToxR form dimers which helps it bind to DNA form dimers which helps it bind to DNA What genes does What genes does ToxRToxR regulate? regulate?

Gene fusions created to constitutive promoter Gene fusions created to constitutive promoter Fusion introduced into strains of Fusion introduced into strains of V. choleraeV. cholerae with with lacZlacZ randomly randomly

inserted around the genomeinserted around the genome Identified intermediate regulator gene Identified intermediate regulator gene ToxTToxT, a transcriptional activator , a transcriptional activator

that binds to promoters of many genes, including that binds to promoters of many genes, including ctxctx ToxR/S or ToxT can activate the ctx genes that produce toxinToxR/S or ToxT can activate the ctx genes that produce toxin ToxT alone activates additional virulence genes which encode pili ToxT alone activates additional virulence genes which encode pili

and other proteinsand other proteins Transcription of ToxT is regulated by ToxR/STranscription of ToxT is regulated by ToxR/S

Fig. 16.22

Unanswered Questions RemainUnanswered Questions Remain

Why is there a cascade (ToxR and ToxT) of Why is there a cascade (ToxR and ToxT) of regulatory factors?regulatory factors?

What DNA sequence in the promoters does What DNA sequence in the promoters does ToxR recognize?ToxR recognize?

What is the signal that’s makes the cholera What is the signal that’s makes the cholera bacteria start to colonize the small bacteria start to colonize the small intestine?intestine?

How does ToxR regulatory protein find How does ToxR regulatory protein find binding sites on the chromosome?binding sites on the chromosome?