chapter15 Gene regulation in prokaryotes-5E1skgjx.whu.edu.cn/Public/upfile/article/201706221716086195.pdf · 15.1 The elements of prokaryotic gene expression 15.2 Regulation of transcription

17-1Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display

Gene Regulation in ProkaryotesGene Regulation in Prokaryotes

Chapter 15Chapter 15


Sections to studySections to study

15.1 The elements of prokaryotic gene expression15.1 The elements of prokaryotic gene expression15.2 Regulation of transcription initiation via15.2 Regulation of transcription initiation via

DNADNA--binding proteinsbinding proteins15.3 RNA15.3 RNA--mediated mechanisms of gene regulationmediated mechanisms of gene regulation15.4 Discovering and manipulating bacterial gene regulatory

mechanisms


Bacteria respond to environmental changes byBacteria respond to environmental changes bychanging gene expressionchanging gene expression

Vibrio cholerae causes cholera


15.1 The elements of prokaryotic gene expression15.1 The elements of prokaryotic gene expression RNA polymeraseRNA polymerase and the three phases of and the three phases of transcriptiontranscription..

InitiationInitiation –– sigma subunit + core enzymesigma subunit + core enzyme Binds to promoter, unwinds DNA, begins polymerization of basesBinds to promoter, unwinds DNA, begins polymerization of bases

ElongationElongation –– core enzyme moves away from promoter, sigma subunit core enzyme moves away from promoter, sigma subunit released, polymerization of released, polymerization of ribonucleotidesribonucleotides..


TerminationTermination RhoRho--dependent terminationdependent termination –– Rho factor recognizes sequence in Rho factor recognizes sequence in

mRNA, binds to it, and pulls it away from RNA polymerase.mRNA, binds to it, and pulls it away from RNA polymerase.

RhoRho--independent terminationindependent termination –– stemstem--loop structure formed by loop structure formed by sequence of 20 bases with a run of 6 or more Usequence of 20 bases with a run of 6 or more U’’s signals release of s signals release of RNA polymerase.RNA polymerase.


PolycistronicPolycistronic mRNAmRNA: One mRNA contains the information of : One mRNA contains the information of several genes.several genes.

TranslationTranslation in prokaryotes begins before in prokaryotes begins before transcriptiontranscription ends! ends! Initiation sites for translation signal Initiation sites for translation signal ribosomesribosomes to bind near 5to bind near 5’’ end of end of

mRNA while downstream transcription is still occurring.mRNA while downstream transcription is still occurring. RibosomesRibosomes can initiate translation at several positions along a single can initiate translation at several positions along a single

mRNA.mRNA.


Transcription initiationTranscription initiation

Shift from initiation to elongationShift from initiation to elongation

Release of mRNA at terminationRelease of mRNA at termination

Stability of mRNAStability of mRNA

Efficiency of Efficiency of ribosomesribosomes to recognize translation initiation sitesto recognize translation initiation sites

Stability of polypeptide productStability of polypeptide product

The regulation of gene expression can occur at many stepsThe regulation of gene expression can occur at many steps


15.2 Regulation of transcription initiation via15.2 Regulation of transcription initiation viaDNADNA--binding proteinsbinding proteins

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

Jacques Monod's “bi-phasic”growth curve


Bacteria grown with two different sugars often displayed two Bacteria grown with two different sugars often displayed two phases of growth. phases of growth.

Lactose

Glucose

Phase IUse glucose

Phase IIUse lactose


The presence of lactose The presence of lactose inducesinduces expression of expression of the genes required for the genes required for lactose utilizationlactose utilization InductionInduction: A process by : A process by

which a signal induces which a signal induces expression of a gene or expression of a gene or set of genes.set of genes.

InducerInducer: The molecule : The molecule responsible for the responsible for the induction of gene induction of gene expression.expression.

Fig 15.2


Liquid --galactosidasegalactosidaseassay using ONPG substrate

Plate --galactosidasegalactosidaseassay using X-Gal substrate

Advantages of Advantages of E. coliE. coli and lactose utilization systemand lactose utilization system Culture large numbers of bacteria allow isolation of rare mutantCulture large numbers of bacteria allow isolation of rare mutants.s. Lactose genes are not essential for survival.Lactose genes are not essential for survival. Induction increases protein level 1000Induction increases protein level 1000--fold making mutant identification fold making mutant identification

easy.easy. Color changes using Color changes using --galactosidasegalactosidase substrates (e.g., ONPG, Xsubstrates (e.g., ONPG, X--

Gal) make measurement of expression levels efficient.Gal) make measurement of expression levels efficient.


François Jacob

Jacques Monod

André Lwoff

Working in the Pasteur Institute in Paris

The 1965 Nobel Prize for Physiology or MedicineThe 1965 Nobel Prize for Physiology or Medicine

Revealed coordinate repression and induction of three genes by Revealed coordinate repression and induction of three genes by studying lactosestudying lactose--utilization mutants.utilization mutants.

Proposed Proposed the the OperonOperon TheoryTheory of gene regulation.of gene regulation.


Identification of lactose-utilization genes

Jacques Monod and his collaborators isolated many LacJacques Monod and his collaborators isolated many Lacmutants unable to grow on lactose.mutants unable to grow on lactose.

Three genes, Three genes, lacZlacZ, , lacYlacY, and , and lacAlacA,, were identified in a tightly were identified in a tightly linked cluster.linked cluster.

Fig. 15.4


Experimental evidence for a repressor proteinExperimental evidence for a repressor protein

Synthesize Synthesize --galactosidasegalactosidase and and laclac permeasepermease all the time, even all the time, even in the absence of inducer.in the absence of inducer. Constitutive mutantsConstitutive mutants: M: Mutants that synthesize certain enzyme all the utants that synthesize certain enzyme all the

time, irrespective of environmental conditions.time, irrespective of environmental conditions.

Suggest thatSuggest that lacIlacI encodes a encodes a repressor repressor –– cells would need cells would need lacIlacIprotein product to prevent expression of protein product to prevent expression of lacZlacZ and and lacYlacY in the in the absence of inducer.absence of inducer.

Mutants of Mutants of lacIlacI gene:gene:


PaJaMoPaJaMo experimentexperiment

Start with a Start with a lacIlacI lacZlacZ

mutant.mutant. Transfer Transfer lacIlacI++ and and lacZlacZ++

genes into the cells on a genes into the cells on a piece of DNA.piece of DNA.

--galactosidasegalactosidase was was detected immediately after detected immediately after DNA transfer, and the DNA transfer, and the synthesis stopped in an synthesis stopped in an hour.hour.

If add lactose, If add lactose, --galactosidasegalactosidase keeps keeps accumulating.accumulating.

Fig. 15.5


Explanation of Explanation of experimental results:experimental results:

Initial lack of repressor Initial lack of repressor allowed synthesis of allowed synthesis of LacZLacZenzyme. As enzyme. As LacILacI is is produced, produced, LacZLacZ synthesis synthesis was shut off, unless an was shut off, unless an inducer is present to inducer is present to prevent repressor from prevent repressor from repressing.repressing.

Conclusion:lacI encodes the negative regulator of the lac genes.


Mutational and structural analysis of the repressorMutational and structural analysis of the repressor

Fig. 15.7, 15.8


Changes in the operator DNA to which the repressorChanges in the operator DNA to which the repressorbinds can also affect repressor activitybinds can also affect repressor activity

Fig. 15.7


The The operonoperon hypothesis:hypothesis:

AA repressorrepressor stops transcription by binding to an hypothetical stops transcription by binding to an hypothetical operatoroperator site near promoter of lactosesite near promoter of lactose--utilization genes.utilization genes.

An An inducerinducer can bind to the repressor and prevents it from can bind to the repressor and prevents it from binding to the operator.binding to the operator.

The repressor (The repressor (allostericallosteric proteinprotein) can change shape when ) can change shape when exposed to exposed to inducerinducer. Repressor bound to inducer can. Repressor bound to inducer can’’t bind t bind to DNA.to DNA.


The The operonoperon theorytheory: A single signal can simultaneously regulate : A single signal can simultaneously regulate the expression of several genes that are clustered together on athe expression of several genes that are clustered together on achromosome and are involved in the same biological process.chromosome and are involved in the same biological process.

OperonOperon (操纵子): A unit of DNA composed of several clustered A unit of DNA composed of several clustered genes, plus a promoter and/or operator. These genes are genes, plus a promoter and/or operator. These genes are regulated in the same way in response to environmental regulated in the same way in response to environmental changes and are transcribed into one single mRNA. changes and are transcribed into one single mRNA.

The genes clustered together on a chromosome are transcribed The genes clustered together on a chromosome are transcribed together as single mRNA (together as single mRNA (polycistronicpolycistronic).).

The The operonoperon theory of gene regulationtheory of gene regulation


The The laclac operonoperon The playersThe players

lacZlacZ, , lacYlacY, , lacAlacA genesgenes that split lactose into glucose and that split lactose into glucose and galactosegalactose

Promoter Promoter (启动子) to which RNA polymerase bindsto which RNA polymerase binds ciscis--acting operator siteacting operator site transtrans--acting repressoracting repressor that can bind to operator (encoded by that can bind to operator (encoded by

lacIlacI gene)gene) InducerInducer that prevents repressor from binding to operatorthat prevents repressor from binding to operator

Fig. 15.4


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

prevents transcription.prevents transcription. Negative regulatory elementNegative regulatory element

Fig. 15.4


InductionInduction

Inducer binds to Inducer binds to repressor.repressor.

Repressor changes Repressor changes shape and can not shape and can not bind to operator.bind to operator.

RNA polymerase RNA polymerase binds to promoter binds to promoter and initiates and initiates transcription of transcription of polycistronicpolycistronic mRNA.mRNA.

Fig. 15.4


AllolactoseAllolactose, but not , but not lactose, is the true lactose, is the true inducer of the inducer of the laclacoperonoperon..

lacZlacZ mutants mutants express express lacYlacY in the in the presence of IPTG, presence of IPTG, but not in the but not in the presence of lactose.presence of lactose.

Fig. 15.4


Proteins act in Proteins act in transtrans; but DNA sites act only in ; but DNA sites act only in ciscis

CisCis--acting elementsacting elements: Short DNA sequences that constitute the : Short DNA sequences that constitute the control elements adjacent to genes. They can only influence the control elements adjacent to genes. They can only influence the expression of adjacent genes on the same DNA molecule.expression of adjacent genes on the same DNA molecule.

TransTrans--acting elementsacting elements: Genes that code for DNA: Genes that code for DNA--binding binding proteins. Their protein products can diffuse through proteins. Their protein products can diffuse through cytoplasm and act at target DNA sites on any DNA molecule in cytoplasm and act at target DNA sites on any DNA molecule in the cell.the cell.


Three experiments elucidate Three experiments elucidate ciscis-- and and transtrans--actingactingelements using Felements using F’’ plasmid plasmid

lacIlacI++ gene encodes a gene encodes a diffusible element that diffusible element that binds to any operator it binds to any operator it encounters regardless of encounters regardless of chromosomal location.chromosomal location.

LacILacI++ protein acts protein acts inintranstrans..

Fig. 15.9


LacILacIss protein acts protein acts in transin trans

LacILacIss superrepressorsuperrepressor can can not bind inducer.not bind inducer.

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

lacIlacIss mutant encodes a mutant encodes a diffusible element that diffusible element that binds to operator binds to operator regardless of regardless of chromosomal location chromosomal location (trans(trans--acting element).acting element).

Fig. 15.10


lacOlacOcc acts acts in in ciscis

Presence of Presence of lacOlacO++

plasmid does not plasmid does not compensate for compensate for lacOlacOcc

mutation on bacterial mutation on bacterial chromosome.chromosome.

Operator is Operator is ciscis--acting acting elementelement

Fig. 15.11



Biochemical experiments support the Biochemical experiments support the operonoperon hypothesishypothesis

Radioactive tag attached Radioactive tag attached to to laclac repressor.repressor. Repressor from Repressor from lacIlacI++ cells cells

purified and mixed with purified and mixed with operator DNA; operator DNA; cosedimentcosediment..

Repressor from Repressor from lacIlacI++

mixed with mutant mixed with mutant operator DNA; no operator DNA; no cosedimentcosediment..

Fig. 15.12

The The laclac repressor binds to repressor binds to operator DNA.operator DNA.


DNaseDNase I I footprintingfootprinting identifies DNA sequenceidentifies DNA sequenceto which a protein bindsto which a protein binds

Fig. 15.15

Mix radioactive-labeled target DNA with or without repressor.

Partial digestion with DNase I. DNase I can’t digest DNA regions covered with proteins.

Separation of DNA fragments and autoradiography.

17-32Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or displayFig. 15.13

Pink: lac repressor tetramer


Summary of the repressorSummary of the repressor

Repressor not bound to inducer can bind to the operator. Repressor bound to inducer can’t bind to DNA. The binding of repressor to operator keeps RNA polymerase

from recognizing the promoter. The repressor has separate domains corresponding to two

functions. Defects in either domain as well as the presence or absence of

the inducer affect the function of the repressor.


Bacteria grown with two different sugars often displayed two Bacteria grown with two different sugars often displayed two phases of growth. phases of growth.

Monod's "bi-phasic“ growth curve


The lac operon is also regulated by positive control

cAMPcAMP binds to binds to CRPCRP((cAMPcAMP receptor receptor protein) when protein) when glucose is low.glucose is low.

CRPCRP--cAMPcAMP binds to binds to regulatory region.regulatory region.

Enhances activity of Enhances activity of RNA polymerase at RNA polymerase at laclac promoter.promoter.

Fig. 15.19

Positive regulation of the lac operon by CRP-cAMP and catabolite repression by glucose.


Regulation of the Regulation of the laclac operonoperon depends on at least two proteins: depends on at least two proteins: the repressorthe repressor (a negative regulator) and (a negative regulator) and CRPCRP--cAMPcAMP (a (a positive regulator).positive regulator).

Under these conditions, the repressor binds inducer and Under these conditions, the repressor binds inducer and become unable to bind to the operator, while CRPbecome unable to bind to the operator, while CRP--cAMPcAMPbinds to a site near the promoter to assist RNA polymerase in binds to a site near the promoter to assist RNA polymerase in the initiation of transcription.the initiation of transcription.

When glucose is present in the medium, there is little When glucose is present in the medium, there is little cAMPcAMPavailable to bind to CRP and therefore little induction of the available to bind to CRP and therefore little induction of the laclac operonoperon, even if lactose is present., even if lactose is present.

Maximum induction of Maximum induction of laclac operonoperon occurs in media occurs in media containing lactose but lacking glucosecontaining lactose but lacking glucose


Most regulatory proteins in transcription are Most regulatory proteins in transcription are oligomericoligomeric

CRP-cAMP complex: dimer

Contain more than one domain. Bind to each other to form oligomers.


lac repressor: tetramer

Blue: CRP-cAMPPink: lac repressor tetramer

Fig. 15.17, 15.13


How regulatory proteins interact with RNA polymerase?How regulatory proteins interact with RNA polymerase?

RepressorRepressor--binding site and RNA polymerasebinding site and RNA polymerase--binding site binding site overlaps.overlaps.

Negative regulators, e.g. Negative regulators, e.g. laclac repressor, physically block RNA repressor, physically block RNA polymerasepolymerase--binding site.binding site.

Fig. 15.16


Positive regulators establish physical contact with RNA Positive regulators establish physical contact with RNA polymerase enhancing enzymepolymerase enhancing enzyme’’s ability to initiate s ability to initiate transcription.transcription.

Fig. 15.18


65% of the human 65% of the human population today has population today has lactose intolerancelactose intolerance: : drinking milk makes them drinking milk makes them ill, with symptoms ill, with symptoms including cramps (including cramps (腹痛腹痛) ) and bloating (and bloating (腹胀腹胀) .) .

Lactase (Lactase (乳糖酶乳糖酶)) is is required for breaking required for breaking down lactose in milk.down lactose in milk.

Lactase persistence and milk drinking in humans


Humans express lactase Humans express lactase only in infants but not in only in infants but not in adults.adults.

Lactase

Lactase

Lactase

Lactase persistence

Lactase persistenceLactase persistence allows allows adults to drink milk. adults to drink milk. 35% of the humans carry a 35% of the humans carry a

mutation that caused mutation that caused constitutive expression of constitutive expression of lactase (lactase (lactase persistencelactase persistence).).


Lactase persistenceLactase persistenceallows adults to allows adults to drink milk. drink milk. The mutation The mutation

occurred between occurred between 7,0007,000--9,000 years 9,000 years ago among several ago among several dairying dairying communities in communities in places like northern places like northern Europe and eastern Europe and eastern Africa.Africa.

Fig. 20.1


15.3 RNA-mediated mechanisms of gene regulation

1.1. Diverse RNA leader devices act Diverse RNA leader devices act in in ciscis to regulate gene to regulate gene expression.expression. AttenuationAttenuation RiboswitchesRiboswitches

2.2. Regulatory small Regulatory small RNAsRNAs act act in transin trans to regulate the to regulate the translation of mRNAs.translation of mRNAs.

3.3. Genes can also be regulated by antisense Genes can also be regulated by antisense RNAsRNAs..


trpRtrpR gene encodes repressor.gene encodes repressor. Tryptophan functions as a Tryptophan functions as a corepressorcorepressor –– tryptophan binds to tryptophan binds to trptrp repressor repressor

allowing it to bind to operator DNA and inhibit transcription.allowing it to bind to operator DNA and inhibit transcription.

1. Diverse RNA leader devices act in cis to regulategene expression

Fig. 15.20

(1)(1) AttenuationAttenuation in the in the trptrp operonoperon of of E. coliE. coli::



RepressorRepressor--independent regulation of the independent regulation of the trptrp operonoperon

When tryptophan is present,When tryptophan is present, trpRtrpR mutants are not completely mutants are not completely dede--repressed in the expression of repressed in the expression of trptrp genes.genes.

The presence of tryptophan can still inhibit the expression of The presence of tryptophan can still inhibit the expression of trptrpgenes in genes in trpRtrpR mutants. mutants.


Two alternative transcripts lead to different transcriptional Two alternative transcripts lead to different transcriptional outcomes.outcomes. RNA leader sequence (~ 140 bases) can fold in two different stabRNA leader sequence (~ 140 bases) can fold in two different stable le

conformations.conformations.



Attenuation (弱化作用): Control of gene expression by premature termination of transcription.


(2)(2) RiboswitchesRiboswitches: : AllostericAllosteric RNA leaders that bind small molecule RNA leaders that bind small molecule effectors to control gene expression.effectors to control gene expression.

17 different riboswitch aptamers have been identified in the E. coli genome. Aptamer: The region of a riboswitch that can bind an effector and

thereby alter its stem-loop structures as well as those of the expression platform connected to it.

Expression platform: The region of a riboswitch that controls gene expression by altering its stem-loop structures in response to the aptamer configuration.



CspA: E. coli cold shock protein, essential for growth in cold. Only at low temperatures, cspA mRNA is stable and is preferentially

translated. 5’UTR: Forms stem-loop at high temperature.

mRNA half-life:37℃：20 s10℃：30 min

37℃ 10℃

37℃ 10℃

Temperature-sensing mRNAs


At low temperatures, stem-loop structures that occlude the ribosome binding site form. At high temperatures, the stem-loop unzips.

The most well-studied RNA thermometer is found in the rpoH gene in E. coli. This thermosensor upregulates heat shock proteins under high temperatures through 32, a specialized heat-shock sigma factor (Storz G. Genes Dev. 1999, 13: 633-636)

RNA thermometers: RNA leaders in some bacterial mRNAs that regulate translation in response to temperature through stem-loop structure whose stability is temperature-dependent.


2. Regulatory small RNAs act in trans to regulate the translation of mRNAs

Bacterial genomes encode many small RNA molecules (sRNAs, 50-400nt long) that regulate translation in trans by base pairing with mRNAs. Most sRNAs inhibit translation of target mRNAs by base pairing with

the ribosome binding site (RBS). Some sRNAs activate translation of target mRNAs by disrupting the

formation of a stem-loop structure in the leader mRNA that would otherwise block the ribosome binding site.

Some sRNAs influence the expression of target mRNAs by base pairing with mRNA and promoting the degradation of the mRNA by ribonucleases.



3. Genes can also be regulated by antisense RNAs Antisense RNAs: Regulatory RNAs that are complementary in

sequence to the mRNAs they regulate because they are transcribed using the opposite strand of DNA as a template. They range in size from 10-1000 nt.

Some antisense RNAs can block transcription or translation of their target mRNAs. Some could inhibit translation of target mRNAs by base pairing with

the sense mRNA and blocking the ribosome binding site. The double-stranded RNA formed by base pairing between antisense

RNA and the target mRNA can be degraded by ribonucleases. The transcription of antisense RNA can interfere with initiation of

transcription of the sense gene in a yet unknown mechanism.

Fig. 15.24


15.4 Discovering and manipulating bacterial gene 15.4 Discovering and manipulating bacterial gene regulatory mechanismsregulatory mechanisms

Reporter geneReporter gene: A protein: A protein--coding gene whose expression in the coding gene whose expression in the cell is quantifiable by techniques of protein detection.cell is quantifiable by techniques of protein detection.

Use of Use of lacZlacZ gene as reporter of gene expression.gene as reporter of gene expression.

Fig. 15.25


Fusion of reporter gene to Fusion of reporter gene to ciscis--acting regulatory regions acting regulatory regions allows assessment of gene activity by monitoring amount of allows assessment of gene activity by monitoring amount of reporter gene product.reporter gene product.

X-Gal staining of 13.5 days mouse embryo carrying Cecr2-lacZ fusion.


Creating a collection Creating a collection of of lacZlacZ insertions in insertions in

the chromosomethe chromosome

Fig. 15.26


laclac operonoperon regulatory regulatory sequences help produce sequences help produce

protein drugs in bacteriaprotein drugs in bacteria

Fig. 15.27


RNARNA--SeqSeq is a general tool for characterizingis a general tool for characterizingtranscriptomestranscriptomes and their regulationand their regulation

TranscriptomeTranscriptome: All the mRNAs expressed in a single cell or : All the mRNAs expressed in a single cell or organism.organism.

RNA-Seq: Method for analysis of the transcriptome of an organism in which millions of cDNAs are sequenced. Each read is about

150 nt long.

Documents

chapter15 Gene regulation in prokaryotes-5E1skgjx.whu.edu.cn/Public/upfile/article/201706221716086195.pdf · 15.1 The elements of prokaryotic gene expression 15.2 Regulation of transcription