《分子生物学》课件CHAPTER16ff

1、1Molecular Biology of the Gene, 5/E - Watson et al. (2004)Part I: Chemistry and Genetics Part II: Maintenance of the Genome Part III: Expression of the GenomePart IV: RegulationPart V: Methods2005-5-10The revised central dogmaRNA processingGene regulation基因组的保持基因组的表达The structure of DNA and RNA3Part

2、 IV RegulationCh 16: Transcriptional regulation in prokaryotesCh 17: Transcriptional regulation in eukaryotesCh18: Regulatory RNAsCh 19: Gene regulation in development and evolutionCh 20: Genome Analysis and Systems Biology4Housekeeping genes: expressed constitutively, essential for basic processes

3、involving in cell replication and growth.Inducible genes: expressed only when they are activated by inducers or cellular factors.Expression of many genes in cells are regulated5Surfing the contents of Part IV-The heart of the frontier biological disciplines 6Some of the peoples who significantly con

4、tribute to the knowledge of gene regulation789Chapter 16Gene Regulation in ProkaryotesMolecular Biology Course10TOPIC 1 Principles of Transcriptional Regulation watch the animation TOPIC 2 Regulation of Transcription Initiation: Examples from Bacteria (Lac operon, alternative s factors, NtrC,MerR, G

5、al rep, araBAD operon)TOPIC 3 The Case of Phage : Layers of Regulation11Topic 1: Principles of Transcription Regulation CHAPTER 16 Gene Regulation in ProkaryotesWhat are the regulatory proteins?Which steps of gene expression to be targeted?How to regulate? (recruitment, allostery, blocking, action a

6、t a distance, cooperative binding)121. 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

7、or repressors DECREASE or ELIMINATE the transcriptionPrinciples of Transcription Regulation132. Most activators and repressors act at the level of transcription initiation Principles of Transcription RegulationWhy that?Transcription initiation is the most energetically efficient step to regulate. A

8、wise decision at the beginningRegulation at this step is easier to do well than regulation of the translation initiation.14Regulation also occurs at all stages after transcription initiation. Why?Allows more inputs and multiple checkpoints. The regulation at later stages allow a quicker response.15F

9、ig 12-3-initiationPromoter Binding (closed complex)Promoter “melting” (open complex)Promoter escape/Initial transcription16Fig 12-3-Elongation and terminationTerminationElongation173. Targeting promoter binding: Many promoters are regulated by activators (激活蛋白) that help RNAP bind DNA (recruitment)

10、and by repressors (阻遏蛋白) that block the binding.Principles of Transcription Regulation18Generally, 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

11、 transcription. 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.19a. Absence of Regulatory Proteins: basal level expressionb. Repressor binding to t

12、he operator repressesexpressionc. Activator binding activates expressionFig 16-1204 Targeting transition to the open complex: Allostery regulation (异构调控) after the RNA Polymerase BindingPrinciples of Transcription RegulationIn some cases, RNAP binds the promoters efficiently, but no spontaneous isom

13、erization (异构化) occurs to lead to the open complex, resulting 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 e

14、xample of allostery regulation.21Fig 16-2Allostery regulation Allostery is not only a mechanism of gene activation , it is also often the way that regulators are controlled by their specific signals.22Repressors can work in ways:blocking the promoter binding. blocking the transition to the open comp

15、lex.blocking promoter escape23 5. Action at a Distance and DNA Looping. The regulator proteins can function even binding at a DNA site far away from the promoter region, through protein-protein interaction and DNA looping.Fig 16-3Principles of Transcription Regulation24Fig 16-4 DNA-binding protein c

16、an facilitate interaction between DNA-binding proteins at a distanceFig 16-4Architectural protein256. Cooperative binding (recruitment) and allostery have many roles in gene regulationFor example: group of regulators often bind DNA cooperatively (activators and/or repressors interact with each other

17、 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).Principles of Transcription Regulation26Watch the animation-regulation of

18、 the transcription initiation!27Topic 2: Regulation of Transcription Initiation : Examples from BacteriaCHAPTER 16 Gene Regulation in Prokaryotes28Operon: a unit of prokarytoic gene expression and regulation which typically includes: 1. Structural genes for enzymes in a specific biosynthetic and met

19、abolic pathway whose expression is coordinately controlled. 2. 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. 29Control elementStructural genes30First example: Lac operonRe

20、gulation of Transcription Initiation in Bacteria The lactose Operon (乳糖操纵子)31Point 1: Composition of the Lac operon 32The 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

21、 carbon source. Fig 16-5The LAC operon1. Lactose operon contains 3 structural genes and 2 control elements.33lacYencodes 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 thioga

22、lactoside transacetylase (硫代半乳糖苷转乙酰酶)to get rid of the toxic thiogalacosides The LAC operon34The 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 site Olac position betwe

23、en bases -5 and +21 at the 3-end of PlacBinds with the lac repressor The LAC operon35Point 2: Regulatory proteins and their response to extracellular signals362. An 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 (

24、cAMP Receptor Protein,cAMP受体蛋白); responses to the glucose level.The repressor: lac repressor that is encoded by LacI gene; responses to the lactose.Sugar switch-off mechanismThe LAC operon373. The activity of Lac repressor and CAP are controlled allosterically by their signals. Lactose is converted

25、to allolactose by b-galactosidase, therefore lactose can indirectly turn off the repressor. Glucose lowers the cellular cAMP level, therefore, glucose indirectly turn off CAP.The LAC operonAllolactose binding: turn of Lac repressorcAMP binding: turn on CAP38Fig 16-6The LAC operon39ipozyaVery low lev

26、el of lac mRNAAbsence of lactoseActiveipozyab-GalactosidasePermeaseTransacetylasePresence of lactoseInactiveLack of inducer: the lac repressor block all but a very low level of trans-cription of lacZYA .When Lactose is present, the low basal level of permease allows its uptake, and b-galactosidase c

27、atalyzes the conversion of some lactose to allolactose.Allolactose acts as an inducer, binding to the lac repressor and inactivate it. Response to lactose40Response to glucose: 注意该图的CRP结合位点有误 41Point 3: The mechanism of the binding of regulatory proteins to their sites42The LAC operonRepressor bindi

28、ng physically prevents RNAP from binding to the promoter, because the site bound by lac repressor is called the lac operator (Olac ), and the Olac overlaps promoter (Plac). 4. CAP and Lac repressor have opposing effects on RNA polymerase binding to the promoterThe LAC operon43The LAC operonCAP binds

29、 to a site upstream of the promoter, and helps RNA polymerase binds to the promoter by physically interacting with RNAP. This cooperative binding stabilizes the binding of polymerase to Plac. The LAC operon44Fig 16-8The LAC operon45The LAC operon5. CAP interacts with the CTD domain of the a-subunit

30、of RNAP The LAC operon46CAP site has the similar structure as the operator, which is 60 bp upstream of the start site of transcription.CAP interacts with the CTD domain of the a-subunit of RNAP and thus promotes the promoter binding by RNAP.Fig 16-9a CTD: C-terminal domain of the a subunit of RNAP47

31、CAP binds as a dimera CTDFig 16-10. CAP has separate activating and DNA-binding surfaceThe LAC operon486. CAP and Lac repressor bind DNA using a common structural motif: helix-turn-helix motif The LAC operonFig 16-11One is the recognition helix that can fits into the major groove of the DNA. Another

32、 one sits across the major grove and makes contact with the DNA backbone.49DNA binding by a helix-turn-helix motifFig 16-12 Hydrogen Bonds between l repressor and the major groove of the operator. 50Lac operon contains three operators: the primary operator and two other operators located 400 bp down

33、stream and 90 bp upstream. Lac repressor binds as a tetramer (四聚体), with each operator is contacted by a repressor dimer (二聚体). respectively. Fig 16-13DNA looping51A regulator (CAP) works together with different repressors at different genes, this is an example of Combinatorial Control.In fact, CAP

34、acts at more than 100 genes in E.coli, working with an array of partners.7: Combinatorial Control (组合调控): CAP controls other genes as well.52Second example: Alternative s factor Regulation of Transcription Initiation in Bacteria Alternative s factors (可变s因子) direct RNA polymerase to alternative prom

35、oters.53 factor subunit bound to RNA polymerase for transcription initiation (Ch 12) Fig 12-7 s and a subunits recruit RNA pol core enzyme to the promoter54 Different factors binding to the same RNAP, conferring each of them a new promoter specificity. 70 factors is the most common one in E. coli un

36、der the normal growth condition. 55Many 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 subtilisBacteriophage factors56Heat sh

37、ock (热休克) Around 17 proteins are specifically expressed in E. coli when the temperature is increased above 37C.These proteins are expressed through transcription by RNA polymerase using an alternative factor 32 coded by rhoH gene. 32 has its own specific promoter consensus sequences.Alternative s fa

38、ctors 57Many bacteriophages synthesizetheir own factors to endow thehost RNA polymerase with a different promoter specificity and hence to selectively express their own phage genes . BacteriophagesAlternative s factors 58B. subtilis SPO1 phage expresses a cascade of factors which allow a defined seq

39、uence of expression of different phage genes.Fig 16-14Alternative s factors 59Third example: NtrC and MerR use allosteric activationRegulation of Transcription Initiation in Bacteria Transcriptional activators NtrC and MerRwork by allostery rather than by recruitment. 60ReviewThe majority of activat

40、ors work by recruitment, such as CAP. These activators simply 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 allo

41、steric change in that complex to activate transcription.611. NtrC has ATPase activity and works at DNA sites far away from the gene.NtrC and MerR and allosteric activationNtrC controls expression of genes involved in nitrogen metabolism (氮代谢), such as the glnA gene.NtrC has separate activating and D

42、NA-binding domains, and binds DNA only when the nitrogen levels are low.62Low nitrogen levels (低水平氮)NtrC phosphorylation and conformational change NtrC (?) binds DNA sites at -150 bp position as a dimer (?)NtrC interacts 54 in RNAP bound to the glnA promoter NtrC ATPase activity provides energy need

43、ed to induce a conformation change in RNAP transcription STARTsFig 16-15 activation by NtrC 632. MerR activates transcription by twisting promoter DNANtrC and MerR and allosteric activationMerR controls a gene called merT, which encodes an enzyme that makes cells resistant to the toxic effects of me

44、rcury (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.64As a 70 promoter, merT contains 19 bp between 10 and 35 elements (the typical length is 15-17 bp), leaving these two elements recognized by 70 neither op

45、timally separated nor aligned. 65Fig 16-15 Structure of a merT-like promoter 66Fig 16-15When Hg2+ is absent, MerR binds to the promoter and locks it in the unfavorable conformationWhen Hg2+ is present, MerR binds Hg2+ and undergoes conformational change, which twists the promoter to restore it to th

46、e structure close to a strong 70 promoter67Repressors work in many ways-reviewBlocking RNA polymerase binding through binding to a site overlapping the promoter. Lac repressorBlocking the transition from the closed to open complex. Repressors bind to sites beside a promoter, interact with polymerase

47、 bound at that promoter and inhibit initiation. E.coli Gal repressorBlocking the promoter escape. P4 protein interaction with PA2c (bacteriophage f29 )68Fourth example: araBAD operonRegulation of Transcription Initiation in Bacteria 691. AraC and control of the araBAD operon by anti-activationThe ar

48、aBAD operonThe promoter of the araBAD operon from E. coli is activated in the presence of arabinose (阿拉伯糖) and the absence of glucose and directs expression of genes encoding enzymes required for arabinose metabolism. This is very similar to the Lac operon. 70Different from the Lac operon, two activ

49、ators AraC and CAP work together to activate the araBAD operon expressionFig 16-18CAP site194 bpDNA looping71Because the magnitude of induction of the araBAD promoter by arabinose is very large, the promoter is often used in expression vector. If fusing a gene to the araBAD promoter, the expression

50、of the gene can be easily controlled by addition of arabinose（阿拉伯糖）.What is an expression vector ? The answer is in the Methods part.72Topic 3: The Case of Bacteriophage l: Layers of RegulationCHAPTER 16 Gene Regulation in Prokaryotes Bacteriophage l is a virus that infects E. coli. Upon infection,

51、the phage can propagate in either of two ways : lytically or lysogenically.The phage l has a 50-kb genome and 50 genes. Most of these genes encode protein for replication, packing or lysis. How the lytic and lysogenic growth is regulated? -regulatory proteins and cis-acting control elements.Alternat

52、ive patterns of gene expression control lytic and lysogenic growth. Fig. 16-21: Promoters in the right and left control regions of phage lFig. 16-22: Transcription in the l control regions in lytic and lysogenic growth2. Regulatory Proteins and Their Binding SitesThe cI gene encodes l repressor, tha

53、t can both activate and repress transcriptionAs a repressor: similarly as Lac repressor (?)As an activator: similarly as CAP (?) l repressorAs a repressor, it binds to sites that overlap the promoter and excludes RNA polymeraseAs an activator, it works like CAP by recruitment.Cro (another regulatory

54、 protein), stands for control of repressor and other things. It is a single domain protein that binds as a dimer to 17-bp DNA sequences using a HTH motif. It only represses transcription. repressor and Cro can each bind to any one of six operators, but with dramatically different affinity. repressor

55、 binds OR1 most easily while Cro binds OR3 with highest affinity.l repressor binds OR1 tenfold better than OR2. Cro binds OR3 tenfold better than OR1 and OR2.There are 6 operators in the right (3) and left (3) control regions of bacteriophage l. Sequences are not identical 3. l repressor binds to op

56、erator sites cooperatively. Two dimmers of repressor bind cooperatively to OR1 and OR2, The binding at OR1 helps the binding at OR2.MonomerDimerTetramerHigh affinity10-fold low affinityNot boundBox 16-3 Concentration, affinity, and cooperative bindingTwo factors determine whether two interacting mol

57、ecules find and bind each others: (1) the binding affinity (2) their concentrations. Cooperativity binding can be expressed in terms of increased affinity. The curve of l repressor binding to its operator DNA.Binding of a protein to a single site82The benefit of cooperative binding of regulatory pro

58、teins is to ensure dramatic changes in the expression level of a given gene even in response to small changes in the level of the control signal.Lytic growthLysogen4. Repressor and Cro bind in different patterns to control lytic and lysogenic growthDNA damage activates RecA in E. coli RecA stimulate

59、s l repressor to undergo autocleavage, resulting in the removal the C-terminal domain and the immediate loss of dimerization and binding cooperativity. Repressor dissociates from OR1-OR2 & OR1-OR2, which triggers transcription from PR and PLleading to lytic growth.5. Lysogenic induction requires pro

60、teolytic cleavage of l repressor85For induction to work efficiently, the level of repressor in a lysogen must be tightly regulated. How?Keep it not too low by positive autoregulation: l repressor binding at OR2 activates its own transcription from PRM. Keep it not too high by negative autoregulation