分子生物学教学课件：Part 4 Chapter 16 Gene Regulation in Prokaryotes

1、1Chapter 16Gene Regulation in ProkaryotesMolecular Biology Course2TOPIC 1 Principles of Transcriptional RegulationTOPIC 2 Regulation of Transcription Initiation: Examples from BacteriaTOPIC 3 The Case of Phage : Layers of Regulation3CHAPTER 16 Gene Regulation in ProkaryotesTopic 1: Principles of Tra

2、nscription Regulation 41. Gene Expression is Controlled by Regulatory Proteins (调控蛋白)Gene expression is very often controlled by Extracellular Signals, which are communicated to genes by regulatory proteins:Positive regulators or activators: INCREASE the transcriptionNegative regulators or repressor

3、s: DECREASE or ELIMINATE the transcription52. Most activators and repressors act at the level of transcription initiation Why that?Transcription initiation is the most energetically efficient step to regulate. A wise decision at the beginningRegulation at this step is easier to do well than regulati

4、on of the translation initiation.6Regulation also occurs at all stages after transcription initiation. Why?Allows more inputs and multiple checkpoints.The regulation at later stages allow a quicker response.73. Targeting promoter binding: Many promoters are regulated by activators (激活蛋白) that help R

5、NAP bind DNA (recruitment) and by repressors (阻遏蛋白) that block the binding.8Generally, RNAP binds many promoters weakly. Why? Activators contain two binding sites to bind a DNA sequence and RNAP simultaneously, can therefore enhance the RNAP affinity with the promoters and increases gene transcripti

6、on. This is called recruitment regulation (招募调控).* On the contrary, Repressors can bind to the operator inside of the promoter region, which prevents RNAP binding and the transcription of the target gene.9a. Absence of Regulatory Proteins: basal level expressionb. Repressor binding to the operator r

7、epressesexpressionc. Activator binding activates expression104. Targeting transition to the open complex: Allostery regulation (异构调控) after the RNA Polymerase BindingIn some cases, RNAP binds the promoters efficiently, but no spontaneous isomerization (异构化) occurs to lead to the open complex, result

8、ing in no or low transcription. Some activators can bind to the closed complex, inducing conformational change in either RNAP or DNA promoter, which converts the closed complex to open complex and thus promotes the transcription. This is an example of allostery regulation.11Allostery regulation Allo

9、stery is not only a mechanism of gene activation , it is also often the way that regulators are controlled by their specific signals. 5. Action at a Distance and DNA Looping:远程激活和DNA环化The regulator proteins can function even binding at a DNA site far away from the promoter region, through protein-pr

10、otein interaction and DNA looping.13Fig 16-4 DNA-bending protein can facilitate interaction between DNA-binding proteins at a distance146. Cooperative binding and allostery have many roles in gene regulation协同结合和变构在基因调控中有多种作用For example: group of regulators often bind DNA cooperatively (activators a

11、nd/or repressors interact with each other and with the DNA, helping each other to bind near a gene they regulated) : Produce sensitive switches to rapidly turn on a gene expression. (1+12)Integrate signals (some genes are activated when multiple signals are present).15CHAPTER 16 Gene Regulation in P

12、rokaryotesTopic 2: Regulation of Transcription Initiation : Examples from Bacteria16Operon: a unit of prokaryotic gene expression and regulation which typically includes: 1. Structural genes for enzymes in a specific biosynthetic and metabolic pathway whose expression is coordinately controlled. 2.

13、Control elements, such as operator sequence. 3. Regulator gene(s) whose products recognize the control elements. These genes is usually transcribed from a different promoter. 操纵子是DNA分子内由一定的核苷酸序列组成的一个基因表达的协同单位，包括几个结构基因及基因表达的共同的调控序列。 17Control elementStructural genes18Regulation of Transcription Initi

14、ation in Bacteria First example: The lactose Operon 乳糖操纵子Diauxic Growth（二次生长曲线）20Composition of the Lac operon 21The enzymes encoded by lacZ, lacY, lacA are required for the use of lactose as a carbon source. These genes are only transcribed at a high level when lactose is available as the sole carb

15、on source. Lactose operon contains 3 structural genes and 2 control elements.22lacYencodes a cell membrane protein called lactose permease (半乳糖苷渗透酶) to transport Lactose across the cell walllacZcodes for -galactosidase (半乳糖苷酶) for lactose hydrolysislacA encodes a thiogalactoside transacetylase (硫代半乳

16、糖苷转乙酰酶)to get rid of the toxic thiogalacosides The b-Galactosidase Reaction乳糖 半乳糖 葡萄糖24The lacZ, lacY, lacA genes are transcribed into a single lacZYA mRNA (polycistronic mRNA) under the control of a single promoter Plac . LacZYA transcription unit contains an operator（操作子） site Olac position betwee

17、n bases -5 and +21 at the 3-end of PlacBinds with the lac repressor 25Expression of the Lac GenesControl of the lac OperonThe lac operon is tightly controlled, using 2 types of controlNegative control, like the brake of a car, must remove the repressor from the operatorAn activator, additional posit

18、ive factor, responds to low glucose by stimulating transcription of the lac operon27An activator and a repressor together control the Lac operon expressionThe activator: CAP (Catabolite Activator Protein,代谢产物激活蛋白) or CRP (cAMP Receptor Protein,cAMP受体蛋白); responses to the glucose level.The repressor:

19、 lac repressor that is encoded by LacI gene; responses to the lactose.Sugar switch-off mechanismThe LAC operonNegative Control of the lac OperonNegative control indicates that the operon is turned on unless something turns it off and stops itThe off-regulation is done by the lac repressor Product of

20、 the lacI geneTetramer of 4 identical polypeptidesBinds the operator just right of promoterlac RepressorWhen repressor binds the operator, operon is repressedOperator and promoter are contiguousRepressor bound to operator prevents RNA polymerase from binding to the promoterAs long as no lactose is a

21、vailable, lac operon is repressedNegative Control of the Lac OperonInducer of the lac OperonThe repressor is an allosteric protein （变构蛋白）Binding of one molecule to the protein changes shape of a remote site on that proteinAltering its interaction with a second moleculeInducer (one molecule) of lac o

22、peron binds the repressorCausing the repressor to change conformation that favors release from the operator (the second molecule)The inducer is allolactose（异乳糖）, an alternative form of lactose7-32Inducer of the lac OperonInducer (one molecule) of lac operon binds the repressorThe inducer is allolact

23、ose, an alternative form of lactose乳糖 异乳糖Positive Control of lac OperonGlucose present, operon inactive. Why?Catabolite Repression of the lac OperonWhen glucose is present, lac operon is in a relatively inactive state This selection in favor of glucose metabolism and against use of other energy sour

24、ces has long been attributed to the influence of some breakdown product, or catabolite. It is known as catabolite repression（代谢阻遏）.Positive Control of lac OperonPositive control of lac operon by a substance sensing lack of glucose that responds by activating lac promoterThe concentration of nucleoti

25、de, cyclic-AMP, rises as the concentration of glucose dropsCatabolite Activator ProteincAMP added to E. coli can overcome catabolite repression of lac operonAddition of cAMP lead to activation of the lac gene even in the presence of glucosePositive controller of lac operon has 2 parts:cAMPProtein fa

26、ctor is known as:Catabolite activator protein or CAPCyclic-AMP receptor protein or CRPGene encoding this protein is crpThe Mechanism of CAP ActionCAP-cAMP complex binds to the lac promoterMutants whose lac gene is not stimulated by complex had the mutation in the lac promoterMapping the DNA has show

27、n that the activator-binding site（激活因子结合位点） lies just upstream of the promoterBinding of CAP and cAMP to the activator site helps RNA polymerase form an open promoter complexCAP Plus cAMP ActionThe open promoter complex does not form even is RNA polymerase has bound the DNA unless the CAP-cAMP compl

28、ex is also boundRecruitmentCAP-cAMP recruits polymerase to the promoter in two stepsFormation of the closed promoter complexConversion of the closed promoter complex into the open promoter complex1. 乳糖操纵子的结构大肠杆菌的乳糖操纵子含Z、Y及A三个 结构基因，分别编码-半乳糖苷酶、透酶、乙酰基转移酶，此外还有一个操纵序列O、一个启动序列P及一个调节基因。基因编码一种阻遏蛋白，后者与O序 列结合，

29、使操纵子受阻遏而处于转录失活状态。在启动序列P上游还有一个分解（代谢）物基因激活蛋白CAP结合位点，由P序列、O序列和CAP结合位点共同 构成LAC操纵子的调控区，三个酶的编码基因即由同一调控区调节，实现基因产物的协调表达。E.coli的乳糖操纵子2. 阻遏蛋白的负性调节在没有乳糖存在时，乳糖操纵子处于阻遏状态。此时，Lac I基因与O序列结合，故阻断转录启动。阻遏蛋白的阻遏作用并非绝对，偶有阻遏蛋白与O序列解聚。因此，每个细胞中可能会有寥寥数分子半乳糖苷酶、透酶生成。当有乳糖存 在时，乳糖操纵子即可被诱导。真正的诱导剂并非乳糖本身。乳糖经透酶催化、转运进入细胞，再经原先存在于细胞中的少数-半

30、乳糖苷酶催化，转变为别乳 糖。后者作为一种诱导剂分子结合阻遏蛋白，使蛋白构型变化，导致阻遏蛋白与O序列解离、发生转录，使-半乳糖苷酶分子增加1000倍。3. CAP的正性调节分解代谢物基因激活蛋白CAP是同二聚体，在其分子内有DNA结合区及cAMP结合位点。当没有葡萄 糖时，cAMP浓度较高，cAMP与CAP结合，这时CAP结合在乳糖启动序列附近的CAP位点，可刺激RNA转录活性，使之提高50倍；当葡萄 糖存在时，cAMP浓度降低，cAMP与CAP结合受阻，因此乳糖操纵子表达下降。由此可见，对乳糖操纵子来说CAP是正性调节因素，乳糖 阻遏蛋白是负性调节因素。两种调节机制根据存在的碳源性质及水平

31、协调调节乳糖操纵子的表达。4.对调节机制的解释大肠杆菌根据碳源性质选择代谢方式。倘若有葡萄糖存在时，细菌优先选择葡萄糖供应能量。葡萄糖通过降 低cAMP浓度，阻碍cAMP与CAP结合而抑制乳糖操纵子转录，使细菌只能利用葡萄糖。在没有葡萄糖而只有乳糖的条件下，阻遏蛋白与O 序列解聚，CAP结合cAMP后与乳糖操纵子的CAP位点，激活转录，使得细菌利用乳糖作为能量来源。44Regulation of Transcription Initiation in Bacteria Alternative s factors (可变s因子) direct RNA polymerase to alternat

32、ive promoters.Second example: Alternative s factor 45 factor subunit bound to RNA polymerase for transcription initiation (Ch 12) s and a subunits recruit RNA pol core enzyme to the promoter46 Different factors binding to the same RNAP, conferring each of them a new promoter specificity. 70 factors

33、is the most common one in E. coli under the normal growth condition. 47Many bacteria produce alternative sets of factors to meet the regulation requirements of transcription under normal and extreme growth condition. Bacteriophage has its own factors E. coli: Heat shock 32 Sporulation in Bacillus su

34、btilisBacteriophage factors48Heat shock (热休克) Around 17 proteins are specifically expressed in E. coli when the temperature is increased to 42C.These proteins are expressed through transcription by RNA polymerase using an alternative factor 32 coded by rhoH gene. 32 has its own specific promoter con

35、sensus sequences.Alternative s factors 49Many bacteriophages synthesize their own factors to endow the host RNA polymerase with a different promoter specificity and hence to selectively express their own phage genes . Bacteriophages50B. subtilis SPO1 phage expresses a cascade of factors which allow

36、a defined sequence of expression of different phage genes.51Transcriptional activators NtrC and MerRwork by allostery rather than by recruitment.以变构而非募集作用的转录活化子 Third example: NtrC and MerR use allosteric activation52The majority of activators work by recruitment, such as CAP. These activators simpl

37、y bring an active form of RNA polymerase to the promoter.The beautiful exceptions: allosteric activation by NtrC and MerR. In allosteric activation RNAP initially binds the promoter in an inactive complex, and the activator triggers an allosteric change in that complex to activate transcription.Revi

38、ew531. NtrC has ATPase activity and works at DNA sites far away from the gene.NtrC controls expression of genes involved in nitrogen metabolism (氮代谢), such as the glnA gene.NtrC has separate activating and DNA-binding domains, and binds DNA only when the nitrogen levels are low.54Low nitrogen levels

39、 (低水平氮)NtrC phosphorylation and conformational change NtrC binds DNA sites at -150 bp position as a dimerNtrC interacts 54 in RNAP bound to the glnA promoter NtrC ATPase activity provides energy needed to induce a conformation change in RNAP transcription STARTs552. MerR activates transcription by t

40、wisting promoter DNAMerR controls a gene called merT, which encodes an enzyme that makes cells resistant to the toxic effects of mercury (抗汞酶)In the presence of mercury (汞), MerR binds to a sequence between 10 and 35 regions of the merT promoter and activates merT expression.56As a 70 promoter, merT

41、 contains 19 bp between 10 and 35 elements (the typical length is 15-17 bp), leaving these two elements recognized by 70 neither optimally separated nor aligned. 57Fourth example: araBAD operonFeatures of the ara OperonTwo ara operators exist:araO1 regulates transcription of a control gene called ar

42、aCaraO2 is located far upstream of the promoter is controlsCAP-binding site is 200 bp upstream of the ara promoter, yet CAP stimulates transcriptionThis operon has another system of negative regulation mediated by the AraC proteinThe ara Operon Repression LoopThe araO2 operator controls transcriptio

43、n from a promoter 250 downstreamDoes the DNA between the operator and the promoter loop out?Robert Lobell and Robert SchleifThe ara Control Protein The AraC, ara control protein, acts as both a positive and negative regulatorThere are 3 binding sitesFar upstream site, araO2araO1 located between -106

44、 and -144araI is really 2 half-sitesaraI1 between -56 and -78 araI2 -35 to -51 Each half-site can bind one monomer of AraCThe araCBAD OperonThe ara operon is also called the araCBAD operon for its 4 genesThree genes, araB, A, and D, encode the arabinose metabolizing enzymesThese are transcribed righ

45、tward from the promoter araPBADOther gene, araCEncodes the control protein AraCTranscribed leftward from the araPc promoter Control of the ara OperonIn absence of arabinose, no araBAD products needed, AraC exerts negative controlBinds to araO2 and araI1Loops out the DNA in betweenRepresses the opero

46、nControl of the ara OperonPresence of arabinose, AraC changes conformationIt can no longer bind to araO2Occupies araI1 and araI2 insteadRepression loop brokenOperon is derepressedAraC Control of the ara OperonIn absence of arabinose, no araBAD products needed, AraC exerts negative controlBinds to ar

47、aO2 and araI1Loops out the DNA in betweenRepresses the operonPresence of arabinose, AraC changes conformationIt can no longer bind to araO2Occupies araI1 and araI2 insteadRepression loop brokenOperon is derepressed66Fifth example: Trp OperonThe trp OperonThe E. coli trp operon contains the genes for

48、 the enzymes the bacterium needs to make the amino acid tryptophanThe trp operon codes for anabolic（合成代谢） enzymes, those that build up a substanceAnabolic enzymes are typically turned off by a high level of the substance producedThis operon is subject to negative control by a repressor when tryptoph

49、an levels are elevatedtrp operon also exhibits attenuationTryptophans Role in Negative Control of the trp OperonFive genes code for the polypeptides in the enzymes of tryptophan synthesisThe trp operator lies wholly within the trp promoterHigh tryptophan concentration is the signal to turn off the o

50、peronPresence of tryptophan helps the trp repressor bind to its operatorNegative Control of the trp Operon无辅基阻遏物单体前导序列衰减子Negative Control of the trp Operon7-71Attenuation in the trp Operon 72Fig 12-10 transcription terminationMechanism of AttenuationAttenuation imposes an extra level of control on a

51、n operon, more than just the repressor-operator systemOperates by causing premature termination of the operons transcript when product is abundantTwo structures available to the leader-attenuator transcriptOverridding Attenuation76CHAPTER 16 Gene Regulation in ProkaryotesTopic 3: The Case of Bacteri

52、ophage l: Layers of RegulationInfection of E. coli by Phage lVirulent phage replicate and kill their host by lysing or breaking it openTemperate phage, such as l, infect cells but dont necessarily killThe temperate phage have 2 paths of reproductionLytic mode: infection progresses as in a virulent p

53、hageLysogenic mode: phage DNA is integrated into the host genomeTwo Paths of Phage ReproductionGenetic Map of Phage lDNA exists in linear form in the phageAfter infection of host begins the phage DNA circularizesThis is possible as the linear form has sticky endsLysogenic ModeA 27-kD phage protein (

54、l repressor, CI) appears and binds to 2 phage operator regionsCI shuts down transcription of all genes except for cI gene for l repressor itselfLysogen（溶源菌）is a bacterium harboring integrated phage DNAThis integrated DNA is called a prophageLytic Reproduction of Phage lLytic reproduction cycle of ph

55、age l has 3 phases of transcription:Immediate earlyDelayed earlyLate Genes of these phases are arranged sequentially on the phage DNA AntiterminationAntitermination is a type of transcriptional switchA gene product serves as antiterminator that permits RNA polymerase to ignore terminators at the end

56、 of the immediate early genesSame promoters are used for both immediate early and delayed early transcriptionLate genes are transcribed when another antiterminator permits transcription of the late genes from the late promoter to continue without premature terminationAntitermination and Transcriptio

57、nOne of 2 immediate early genes is crocro codes for a repressor of cI gene that allows lytic cycle to continueOther immediate early gene is N coding for N, an antiterminatorN Antitermination FunctionGenetic sites surrounding the N gene include:Left promoter, PLOperator, OLTranscription terminatorN u

58、tilization site(N 蛋白利用位点）N Antitermination FunctionTranscription in the absence of NRNA polymerase begins transcribing leftward at PL and stops at the terminator. The N mRNA is the only product of this transcription.N Antitermination FunctionWhen N is present:N binds transcript of N utilization site (nut site)Interacts with protein complex bound to polymerasePolymerase ignores normal transcription terminator, continues into delayed early genesProtein Complexes Invo