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Genetics: From Genes to Genomes

Genetics: From Genes to Genomes

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Powerpoint <strong>to</strong> accompany<strong>Genetics</strong>: <strong>From</strong> <strong>Genes</strong> <strong>to</strong> <strong>Genomes</strong>Third EditionHartwell ● Hood ● Goldberg ● Reynolds ● Silver ●VeresChapter17Prepared by Malcolm SchugUniversity of North Carolina GreensboroCopyright © The McGraw-Hill Companies, Inc. Permission required <strong>to</strong> reproduce or display 17-1Copyright © The McGraw-Hill Companies, Inc. Permission required <strong>to</strong> reproduce or display


Gene Regulation inProkaryotesCopyright © The McGraw-Hill Companies, Inc. Permission required <strong>to</strong> reproduce or display17-2


Outline of Chapter 17• There are many steps in gene expression and regulationcan occur at any one of them.• Genetic and molecular studies show that most regulationaffects the initiation of RNA transcripts.• Studies of genes for lac<strong>to</strong>se utilization• Negative regulation – blocks transcription• Positive regulation – increases transcription• DNA binding proteins acting on RNA polymerase at promoter are mainagents of regulation• Attenuation of expression – tryp<strong>to</strong>phan pathway• Gene expression is fine tuned by premature termination oftranscription.Copyright © The McGraw-Hill Companies, Inc. Permission required <strong>to</strong> reproduce or display17-3


RNA polymerase is the key enzymefor transcription.• RNA polymerase involved in three phases oftranscription• Initiation – sigma subunit + core enzyme (two alpha, one beta,and one beta’ subunit)• Binds <strong>to</strong> promoter, unwinds DNA, begins polymerization of basescomplementary <strong>to</strong> DNA template• Elongation – movement away from promoter sigma subunitreleased, polymerization• Termination –signal reached by RNA polymerase• Rho dependent termination – Rho fac<strong>to</strong>r recognizes sequence in mRNA,binds <strong>to</strong> it, and pulls it away from RNA polymerase• Rho independent termination – stem loop structure formed by sequenceof 20 bases with a run of 6 or more U’s U s signals release of RNApolymeraseCopyright © The McGraw-Hill Companies, Inc. Permission required <strong>to</strong> reproduce or display17-5


Fig. 17.2Copyright © The McGraw-Hill Companies, Inc. Permission required <strong>to</strong> reproduce or display17-6


• Translation in prokaryotes starts before transcriptionends.• Initiation sites for translation signal ribosomes <strong>to</strong> bind near 5’ 5end of mRNA while downstream transcription is stilloccurring.• Polycistronic mRNAs often lead <strong>to</strong> the translation of severalgenes at the same time from one mRNA transcript.• The regulation of gene expression can occur at manysteps.• Binding of RNA polymerase <strong>to</strong> promoter• Shift from initiation <strong>to</strong> elongation• Release of mRNA at termination• Posttranscriptional stability of mRNA• Efficiency of ribosomes <strong>to</strong> recognize translation initiation sites• Stability of polypeptide productCopyright © The McGraw-Hill Companies, Inc. Permission required <strong>to</strong> reproduce or display17-7


The utilization of lac<strong>to</strong>se by E. coli:A model system for gene regulation• The presence of lac<strong>to</strong>se induces expression of the genesrequired for lac<strong>to</strong>se utilization• Induction – stimulation of protein synthesis• Inducer – molecule that stimulates synthesis• Lac<strong>to</strong>se – inducer of genes for lac<strong>to</strong>se utilization• 1950s and 1960s – Golden era of bacterial genetics• Advantages of E. coli and lac<strong>to</strong>se utilization system• Culture large numbers of bacteria allow isolation of rare mutants• Lac<strong>to</strong>se genes not essential for survival (can use glucose as carbonsource)• Induction increases expression 1000-fold making mutant identificationeasy• Color changes using β-galac<strong>to</strong>sidaseenzyme (e.g., OPNG, X-gal) Xmakemeasurement of expression levels efficient.Copyright © The McGraw-Hill Companies, Inc. Permission required <strong>to</strong> reproduce or display17-8


Coordinate repression and induction of three genesrevealed by studies of lac<strong>to</strong>se-utilization mutants• Jacques Monod and Francois Jacob –Pasteur Institute in Paris• Proposed Operon Theory of gene regulation• Single signal can simultaneously regulate expressionof several genes that are clustered <strong>to</strong>gether on achromosome and involve the same process.• Because genes are clustered, they are transcribed<strong>to</strong>gether as single mRNA.• Clusters of genes are called Operons.Copyright © The McGraw-Hill Companies, Inc. Permission required <strong>to</strong> reproduce or display17-9


The Operon Theory• The players• lacz, lacY, lacZ genes that split lac<strong>to</strong>se in<strong>to</strong> glucose and galac<strong>to</strong>se• Promo<strong>to</strong>r site <strong>to</strong> which RNA polymerase binds• cis acting opera<strong>to</strong>r site• trans-acting acting repressor that can bind <strong>to</strong> opera<strong>to</strong>r (encoded by lacI gene)• Inducer that prevents repressor from binding <strong>to</strong> opera<strong>to</strong>rFig. 17.5 aCopyright © The McGraw-Hill Companies, Inc. Permission required <strong>to</strong> reproduce or display17-10


The Operon Theory• Repression• In absence of lac<strong>to</strong>se, repressor binds <strong>to</strong> opera<strong>to</strong>r whichprevents transcription.• Negative regula<strong>to</strong>ry elementFig. 17.5 bCopyright © The McGraw-Hill Companies, Inc. Permission required <strong>to</strong> reproduce or display17-11


The Operon Theory• Induction• Lac<strong>to</strong>se present• Allolac<strong>to</strong>se binds <strong>to</strong>repressor.• Repressor changes shapeand can not bind <strong>to</strong>opera<strong>to</strong>r.• RNA polymerase binds<strong>to</strong> promo<strong>to</strong>r and initiatestranscription ofpolycistronic mRNA.Fig. 17.5 cCopyright © The McGraw-Hill Companies, Inc. Permission required <strong>to</strong> reproduce or display17-12


Complementation Analysis of mutants identifieslac<strong>to</strong>se utilization genes.• Monod et al. isolated many Lac - mutants unable <strong>to</strong>utilize lac<strong>to</strong>se.• Complementation analysis identified three genes(lacZ, lacY, , and lacA) ) in a tightly linked cluster.Fig. 17.6Copyright © The McGraw-Hill Companies, Inc. Permission required <strong>to</strong> reproduce or display17-13


Experimental evidence for repressor protein• Isolated mutant in lacI gene:• Constitutive mutant – synthesized β-galac<strong>to</strong>sidaseandlac permease even in absence of lac<strong>to</strong>se (inducer)• lacI must be a repressor – cells must need lacI proteinproduct <strong>to</strong> prevent expression of lacY and lacZ inabsence of inducerCopyright © The McGraw-Hill Companies, Inc. Permission required <strong>to</strong> reproduce or display17-14


PaJaMo experiment• lacI+,lacZ+ + x lacI- lacZ- F-• Initial synthesis of β-galac<strong>to</strong>sidase s<strong>to</strong>ps.• Addition of inducer resumessynthesis.• Conclusion – initial lack ofrepressor allows synthesis.As lacI is transferred,synthesis s<strong>to</strong>ps.• Repressor s<strong>to</strong>pstranscription by binding <strong>to</strong>opera<strong>to</strong>r site near promoter.Fig. 17.7Copyright © The McGraw-Hill Companies, Inc. Permission required <strong>to</strong> reproduce or display17-15


Inducer releases repressor <strong>to</strong> triggerenzyme synthesis.• Addition of lac<strong>to</strong>se inducer caused β –galac<strong>to</strong>sidase synthesis <strong>to</strong> continue.• Conclusion: Inducer binds <strong>to</strong> repressor sorepressor can not bind <strong>to</strong> DNA• Allosteric effect - inducer bound <strong>to</strong> promo<strong>to</strong>rchanges conformation of protein so it can notbind <strong>to</strong> DNACopyright © The McGraw-Hill Companies, Inc. Permission required <strong>to</strong> reproduce or display17-16


Repressor has binding domains foropera<strong>to</strong>r and for the inducer.Fig. 17.8 a-bCopyright © The McGraw-Hill Companies, Inc. Permission required <strong>to</strong> reproduce or display17-17


Changes in the opera<strong>to</strong>r can alsoaffect repressor activity.Fig. 17.9Copyright © The McGraw-Hill Companies, Inc. Permission required <strong>to</strong> reproduce or display17-18


Proteins act in trans;DNA sites act only in cis.• Trans acting elements can diffuse throughcy<strong>to</strong>plasm and act at target DNA sites onany DNA molecule in cell.• Cis acting elements can only influenceexpression of adjacent genes on same DNAmolecule.Copyright © The McGraw-Hill Companies, Inc. Permission required <strong>to</strong> reproduce or display17-19


Three experiments elucidate cis and transacting elements using F’ F plasmid.Insert Figure 17.9ahere• Inducible synthesis• lacI + gene encodes adiffusible element thatacts in trans bybinding <strong>to</strong> anyopera<strong>to</strong>r it encountersregardless ofchromosomal location.Fig. 17.10 aCopyright © The McGraw-Hill Companies, Inc. Permission required <strong>to</strong> reproduce or display17-20


NoninducibleInsert Figure 17.9bhere• All opera<strong>to</strong>r sites (O+)eventually occupied bysuperrepressor• lacI superrepressor cannot bind inducer.• lacI s mutant encodes adiffusible element thatbinds <strong>to</strong> opera<strong>to</strong>rregardless of chromosomallocation (trans actingelement).Fig. 17.10 bCopyright © The McGraw-Hill Companies, Inc. Permission required <strong>to</strong> reproduce or display17-21


ConstitutiveInsert Figure 17.9chere• Presence of O +plasmid does notcompensate for O cmutation on bacterialchromosome.• Opera<strong>to</strong>r is cis actingelementFig. 17.10 cCopyright © The McGraw-Hill Companies, Inc. Permission required <strong>to</strong> reproduce or display17-22


Positive control increasestranscription of lacZ, lacY, , and lacA.• cAMP binds <strong>to</strong>CRP (cAMP(recep<strong>to</strong>r protein)when glucose islow.• CRP binds <strong>to</strong>regula<strong>to</strong>ry region.• Enhances activityof RNApolymerase at lacpromo<strong>to</strong>rFig. 17.11Copyright © The McGraw-Hill Companies, Inc. Permission required <strong>to</strong> reproduce or display17-23


Some positive regula<strong>to</strong>rs increasetranscription of genes in only one pathway.• AraC is a positiveregula<strong>to</strong>r for allarabinose geneswhich break downsugar arabinose.• Loss of functionmutation results inlittle or noexpression of genes.Fig. 17.12Copyright © The McGraw-Hill Companies, Inc. Permission required <strong>to</strong> reproduce or display17-24


Molecular studies help fill in detailsof control mechanisms.• Radioactive tag attached<strong>to</strong> lac repressor• Repressor from lacI + cellspurified and mixed withopera<strong>to</strong>r DNA; cosedimen<strong>to</strong>ccurred• Repressor from lacI + mixedwith mutant opera<strong>to</strong>r DNA;no cosediment occurredFig. 17.13Copyright © The McGraw-Hill Companies, Inc. Permission required <strong>to</strong> reproduce or display17-25


Many DNA-Binding proteins containa helix-turnturn-helix motif.• Two a-helical aregionsseparated by a turn inthe protein structure• Helix-turnturn-helix motiffits in<strong>to</strong> major grooveof DNA• Most repressorproteinsFig. 17.14 aCopyright © The McGraw-Hill Companies, Inc. Permission required <strong>to</strong> reproduce or display17-26


Specific amino acids in the a-helix adeterminethe binding specificity of repressor proteins.Fig. 17.14 bHybrid 434-P22 repressor engineered <strong>to</strong> have amino acidsequence that will bind <strong>to</strong> bacterial virus 434 andbacteriophage P22Copyright © The McGraw-Hill Companies, Inc. Permission required <strong>to</strong> reproduce or display 17-27


Most regula<strong>to</strong>ry proteins are oligomeric.• More than onebinding domain• DNase footprintidentifies bindingregion.• DNase cannotdigest proteincovered sites.Fig. 17.15 aCopyright © The McGraw-Hill Companies, Inc. Permission required <strong>to</strong> reproduce or display17-28


How regula<strong>to</strong>ry proteins interactwith RNA polymerase• Negative regula<strong>to</strong>rs (lac(repressor)• Physically block DNA-binding sites of RNApolymerase• Positive regula<strong>to</strong>rs• Establish physical contact with RNApolymerase enhancing enzyme’s s ability <strong>to</strong>initiate transcriptionCopyright © The McGraw-Hill Companies, Inc. Permission required <strong>to</strong> reproduce or display17-30


Using the lacZ gene as a reporter ofgene expression• Reporter gene – protein encoding genewhose expression in the cell is quantifiableby techniques of protein detection• Fusion of reporter gene <strong>to</strong> cis actingregula<strong>to</strong>ry regions allows assessment geneactivity by moni<strong>to</strong>ring amount of reportergene product.Copyright © The McGraw-Hill Companies, Inc. Permission required <strong>to</strong> reproduce or display17-31


Fusion used <strong>to</strong> perform genetic studies of theregula<strong>to</strong>ry region of gene XFig. 17.18 aCopyright © The McGraw-Hill Companies, Inc. Permission required <strong>to</strong> reproduce or display17-32


Creating acollection oflacZinsertions inthechromosomeFig. 17.18 bCopyright © The McGraw-Hill Companies, Inc. Permission required <strong>to</strong> reproduce or display17-33


Use of a fusion <strong>to</strong>overproduce a geneproductFig. 17.18 cCopyright © The McGraw-Hill Companies, Inc. Permission required <strong>to</strong> reproduce or display17-34


The attenuation of gene expression: Fine tuning the trpoperon through termination of transcription• The presence of tryp<strong>to</strong>phan activates arepressor of the trp operon.• trpR gene produces repressor• Corepressor – tryp<strong>to</strong>phan binds <strong>to</strong> trp repressorallowing it <strong>to</strong> bind <strong>to</strong> opera<strong>to</strong>r DNA and inhibittranscriptionCopyright © The McGraw-Hill Companies, Inc. Permission required <strong>to</strong> reproduce or display17-35


Fig. 17.19Copyright © The McGraw-Hill Companies, Inc. Permission required <strong>to</strong> reproduce or display17-36


Termination of transcription finetunes regulation of trp operon.• trpR - mutants are not constitutive.• Repressor independent change in trp expression• Two alternative transcripts lead <strong>to</strong> differenttranscriptional outcomes.• Leader sequence can fold in two different stableconformations.• Tryp<strong>to</strong>phan present – ribosome moves quickly past codons inleader allowing stem-loop <strong>to</strong> form terminating transcription• Tryp<strong>to</strong>phan absent – ribosome stalls allowing normal stem loopstructure <strong>to</strong> form and transcription proceeds normallyCopyright © The McGraw-Hill Companies, Inc. Permission required <strong>to</strong> reproduce or display17-37


Fig. 17.20Copyright © The McGraw-Hill Companies, Inc. Permission required <strong>to</strong> reproduce or display17-38


Global regula<strong>to</strong>ry mechanisms coordinate theexpression of many genes.• Normal sigma fac<strong>to</strong>r (σ(70 ) binds <strong>to</strong> RNA polymerase andrecognizes sequence in promoter <strong>to</strong> initiate transcription.• Heat shock disables σ 70 .• Product of rpoH gene, σ 32 binds <strong>to</strong> sequence in promoter ofheat shock genes when heat stressed and startstranscription.Fig. 17.21 aCopyright © The McGraw-Hill Companies, Inc. Permission required <strong>to</strong> reproduce or display17-39


Fac<strong>to</strong>rs influencing increase in σ 32activity after heat shock• Increase in transcription of the rpoH gene• Increase in the translation of σ 32 mRNAstemming from greater stability of rpoH mRNA• Increase in the stability and activity of the σ 32protein. Chaperones DnaJ/K bind and inhibit σ 32under normal conditions. At high temperature,binding <strong>to</strong> σ 32 does not occur and more σ 32 is free<strong>to</strong> associate with RNA polymerase.• Inactivity of σ 70 decreases competition with σ 32 <strong>to</strong>form RNA polymerase holoenzyme.Copyright © The McGraw-Hill Companies, Inc. Permission required <strong>to</strong> reproduce or display17-40


What enables transcription of σ 32during heat shock?• Normal temperatures, rpoH gene (encodes σ 3232 ) haspromoter sequence recognized by σ 70 which startstranscription• High temperatures (no σ 70 ) a different promotersequence of the rpoH gene is recognized by a differentsigma fac<strong>to</strong>r, σ 24Figure 17.21 bCopyright © The McGraw-Hill Companies, Inc. Permission required <strong>to</strong> reproduce or display17-41


Summary• E. coli’s heat shock response is controlled byalternative sigma fac<strong>to</strong>rs that recognize differentpromoter sequences.• Alternative sigma fac<strong>to</strong>rs bind <strong>to</strong> RNA polymeraseas temperatures change <strong>to</strong> start transcription ofheat shock proteins.• The induction of alternative sigma fac<strong>to</strong>rs thatrecognize different promoter sequences serve asglobal control regula<strong>to</strong>ry mechanisms in E. coliand many other bacteria.Copyright © The McGraw-Hill Companies, Inc. Permission required <strong>to</strong> reproduce or display17-42


Microarrays – a <strong>to</strong>ol for uncoveringchanges in gene expression• Cellular responses <strong>to</strong> global environmentalchanges can be measured by microarrayanalysis of mRNA isolated from culturesgrown in different environmental conditions.• Comparisons of wild-type cultures withstrains containing mutations in keyregula<strong>to</strong>ry regions help identify genes andregula<strong>to</strong>ry elements involved in response <strong>to</strong>specific environmental changes.Copyright © The McGraw-Hill Companies, Inc. Permission required <strong>to</strong> reproduce or display17-43


Fig. 1.13Copyright © The McGraw-Hill Companies, Inc. Permission required <strong>to</strong> reproduce or display17-44


Regulation of Virulence <strong>Genes</strong> inV. cholerae• Bacterial agents of cholera sense changes inenvironment and transmit signals <strong>to</strong>regula<strong>to</strong>rs that initiate, enhance, diminish,or repress expression of various genes.• Three regula<strong>to</strong>ry proteins – ToxR, ToxS,and ToxT – turn on the genes for virulence.Copyright © The McGraw-Hill Companies, Inc. Permission required <strong>to</strong> reproduce or display17-45


Experiments generate model for regulation ofvirulence genes in V. cholerae.• Cloned two genes encoding subunits ofcholera <strong>to</strong>xin: ctxA and ctxB• Made ctxA-lacZreporter gene fusion• Created vec<strong>to</strong>r library of V. choleraegenomic DNA• Used E. coli <strong>to</strong> perform geneticmanipulationsCopyright © The McGraw-Hill Companies, Inc. Permission required <strong>to</strong> reproduce or display17-46


• Isolated a gene that regulates expression of ctx operon• Transformed E. coli containing ctx-lacZconstruct with clonescontaining V. cholerae DNA• Clones that contain a positive regula<strong>to</strong>r should turn on ctx-lacZconstruct.• Identified ToxR, , a transmembrane protein• Identified ToxS, , helps ToxR form dimers which helps it bind <strong>to</strong> DNA.• What genes does ToxR regulate?• Gene fusions created <strong>to</strong> constitutive promoter• Fusion introduced in<strong>to</strong> strains of V. cholerae with lacZ randomlyinserted around the genome• Identified intermediate regula<strong>to</strong>r gene ToxT, , a transcriptional activa<strong>to</strong>rthat binds <strong>to</strong> promoters of many genes, including ctx• ToxR/S or ToxT can activate the ctx genes that produce <strong>to</strong>xin• ToxT alone activates additional virulence genes which encode piliand other proteins.• Transcription of ToxT is regulated by ToxR/S.Copyright © The McGraw-Hill Companies, Inc. Permission required <strong>to</strong> reproduce or display17-47


Fig. 17.22Copyright © The McGraw-Hill Companies, Inc. Permission required <strong>to</strong> reproduce or display17-48


Unanswered Questions Remain• Why is there a cascade (ToxR(and ToxT) ) ofregula<strong>to</strong>ry fac<strong>to</strong>rs?• What DNA sequence in the promoters doesToxR recognize?• What is the signal that makes the cholerabacteria start <strong>to</strong> colonize the smallintestine?• How does ToxR regula<strong>to</strong>ry protein findbinding sites on the chromosome?Copyright © The McGraw-Hill Companies, Inc. Permission required <strong>to</strong> reproduce or display17-49

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