(乳糖操纵子)

1、1Chapter 16Gene Regulation in ProkaryotesMolecular Biology CourseThere are four major parts in this chapter: * Principles of Transcriptional Regulation * Regulation of Transcription Initiation * Examples of Gene Regulation at Steps after Transcription Initiation * The Case of Phage:Layers of Regulat

2、ionoutlinePart One Principles of Transcriptional RegulationCHAPTER 16 Gene Regulation in Prokaryotes Gene Expression is Controlled by Regulation Proteins: Activators and Repressors1.Activators, or Positive regulators, increase transcription of the regulated gene; Repressors, or negative regulators,

3、decrease or eliminate that transcription.2. Many Promoters Are Regulated by Activation that Help RNA Polymerase Bind DNA and by Repressors that Block that Binding. a. Absence of Regulatory Proteins(operator)b. To RepressExpression c. To Activate ExpressionFig 16-13.Some Activators Work by Allostery

4、and Regulate Steps after RNA Polymerase Binding:In some cases, RNA Polymerase binds efficiently unaided and forms a stable closed complex, which does not spontaneously undergo transition to the open complex.Activator that stimulate this kind of promoter wrk by triggering a conformational change in e

5、ither RNA Polymerase or DNA.That is, they interact with the stable closed complex and induce a conformational change that causes transition to the open complex. This mechanism is an example of allostery.Fig 16-2 Action at a Distance and DNA Looping.Some proteins interact with each other even when bo

6、und to sites well separated on the DNAFig 16-3 DNA-bending protein can facilitate interaction between DNA-binding proteins at a distanceFig 16-4In this example, we also call the DNA-binding protein “architectural” proteins. Cooperative Binding and Allostery have Many Roles in Gene RegulationCooperat

7、ive binding: the activator interacts simultaneously with DNA and polymerase and so recruits the enzyme to the promoter.Two roles: IN RESPONSE TO SMALL CHANGES SENSITIVELY and SERVE TO INTEGRATE SIGNALSAllostery is not only a mechanism of gene activation , it is also often the way that regulators are

8、 controlled by their specific signals. Antitermination and Beyond: Not All of Gene Regulation Targets Transcription InitiationThe bulk of gene regulation takes place at the initiation of transcription in both eukaryotes and bacteria.But regulation is certainly not restricted to that step in either c

9、lass of organism. In this chapter we will see examples, in bacteria, of gene regulation that involve transcriptional elongation, RNA processing, and translation of the mRNA into protein.Part TwoCHAPTER 16 Gene Regulation in ProkaryotesRegulation of Transcription Initiation: Examples From BacteriaEXA

10、MPLE ONE-LAC OPERONFig 16-5Lactose operon: a regulatory gene and 3 stuctural genes, and 2 control elementslacIRegulatory genelacZlacYlacADNAm-RNA -GalactosidasePermeaseTransacetylaseProteinStructural GenesCis-acting elementsPlacIPlacOlacThe LAC operonlacYencodes a cell membrane protein called lactos

11、e 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 (硫代半乳糖苷转乙酰酶)to get rid of the toxic thiogalacosides The LAC operon An Activator and a Repressor Together Control the lac GenesThe act

12、ivator is called CAP( Catabolite Activator Protein ) .CAP can bind DNA and activate the lac genes only in the absence of glucose.The lac repressor can bind DNA and repress transcrition only in the absence of lactose.Both CAP and lac repressor are DNA-binding proteins and each binds to a specific sit

13、e n DNA at or near the lac promoter.Fig 16-6The LAC operon CAP and lac repressor have opposing effects on RNA polymerase binding to the lac promoter -the site bound by lac repressorThis 21 bp sequence is twofold summetric and is recognized by two subunits of lac repressor, one binding to each half-s

14、ite.Fig 16-7 lac operator overlaps promoter, and so repressor bound to the operator physically prevents RNA polymerase from binding to the promoter.Fig 16-8CAP binds as a dimer to a site similar in length to the lac operator, but different in sequence and location.CAP has separate activating and DNA

15、-binding surfaces.Fig 16-9At the promoter,where there is no UP-element, a CTD a CTD binds to CAP and adjacent DNA instead.binds to CAP and adjacent DNA instead.CAP and lac repressor bind DNA using a common structural motif 1.The Same A. The protein binds as a homodimer to a site that is an inverted

16、repeat or near repeat. B.Both CAP and lac repressor bind DNA using a helix-turn-helix motif.One of the two helices in helix-turn-helix domain is the recognition helix that can fits into the major groove of the DNA.Fig 16-11The second helix of the helix-turn-helix domain sits across the major groove

17、an makes contact with the DNA backbone , ensuring proper presentation of the recognition helix, and at the same time adding binding energy to the overall protein-DNA interaction.DNA binding by a helix-turn-helix motifFig 16-12 Hydrogen Bonds between l l repressor and the major groove of the operator

18、 The LAC operon2. The DifferenceLac repressor binds as a tetramer, with each operator is contacted by a repressor dimer.Fig 16-13In some cases, other regions of the protein, outside the helix-turn-helix domain, also interact with the DNA.In many cases, binding of the protein does not alter the struc

19、ture of the DNA .In some cases, however, various distortions are seen in the protein-DNA complex. The activity of Lac repressor and CAP are controlled allosterically by their signalsBinding of the corresponding signals alter the structure of these two regulatory proteinsipozyaVery low level of lac m

20、RNAAbsence of lactoseActiveipozyab b-GalactosidasePermeaseTransacetylasePresence of lactoseInactiveLack of inducer: the lac repressor block all but a very low level of trans-cription of lacZYA .Lactose is present, the low basal level of permease allows its uptake, and-galactosidase catalyzes the con

21、version of some lactose to allolactose.Allolactose acts as an inducer, binding to the lac repressor and inactivate it. Response to lactoseResponse to glucoseThe LAC operon Combinatorial Control: CAP controls other genes as wellThe lac genes provide an example of signal integration: their expression

22、is controlled by two signals, each of which is communicated to the genes via a single regulatorthe lac repressor and CAP, respectively.A regulator (CAP) works together with different repressor at different genes, this is an example of Combinatorial Control. In fact, CAP acts at more than 100 genes i

23、n E.coli, working with an array of partners.vCombinatorial control is a characteristic feature of gene regulation. More complex organismshigher eukaryotes in particular-tend to have more signal integration.EXAMPLE TWO- ALTERNATIVE Alternative s factor direct RNA polymerase to alternative site of pro

24、motersRecall from Chapter 12 that it is the subunit of RNA polymerase that recognizes the promoter suquences.Promoter recognitionDifferent factors binding to the same RNA Pol Confer each of them a new promoter specificity Many bacteria produce alternative sets of factors to meet the regulation requi

25、rements of transcription under normal and extreme growth condition.vHeat shock- 32 When E.coliis subject to heat shock, the amount of this new factor increases in the cell, it displaces 70 from a proportion of RNA polymerases ,and directs those enzymes to transcribe genes whose products protect the

26、cell from the effects of heat shock. The level of 32 is increased by two mechanisms: first, its translation is stimulated-that is,its mRNA is translated with greater efficiency after heat shock than it was before; and second, the protein is transiently stabilized.Many bacteriophages synthesizetheir

27、own factors to endow thehost RNA polymerase with a different promoter specificity and hence to selectively express their own phage genes . BacteriophagesB. subtilis SPO1 phage expresses a cascade of factors which allow a defined sequence of expression of different phage genes .Fig 16-14Normal bacter

28、ial holoenzyme Express early genes Encodefactor for transcription of late genes Encode 28 Express middle genes (gene 34 and 33 ) EXAMPLE THREE-NtrC and MerR NtrC and Mert: Transcriptional Activators that Work by Allostery Rather than by RecruitmentNtrC controls expression of genes involved in nitrog

29、en metabolism, such as the glnA gene. At the glnA gene, Ntrc induces a conformational change in the RNA Polymerase, triggering tansition to the open complex.MerR controls a gene called merT. Like NtrC, MerR induces a conformational change in the inactive RNA polymerase-promoter complex, and this cha

30、nge can trigger open complex formation. NtrC Has ATPase Activity and Works from DNA Sites Far from the GeneNtrC has separate activating and DNA-binding domains, and binds DNA only when the nitrogen levels are low.Fig 16-15 activation by NtrC The major process:Low nitrogen levelsNtrB phosphorylates N

31、trCNtrCs DNA-binding domain revealedNtrC binds four sites located some 150 base pairs upstream of the promoterNtrC interacts with 54ATP hydrolysis and conformation change in polymeraseTrigger polymerase to initiate transcription MerR activates transcription by twisting promoter DNAMerR controls a ge

32、ne called merT, which encodes an enzyme that makes cells resistant to the toxic effects of mercury In the presence of mercury, MerR binds to a sequence between 10 and 35 regions of the merT promoter and activates merT expression.The merT promoter is unusual. The distance between the -10 and -35 elem

33、ents is 19bp instead of the 15 to 17 bp typically found in an eddicient 70 promoter. So, these two elements recognized by are neither optimally seperated nor aligned.Fig 16-15 aThe binding of MerR locks the promoter in the unpropitious conformation in the absence of Hg2+ .Fig 16-15 bWhen Hg2+ is pre

34、sent, MerR binds Hg2+ and undergo conformational change, which twists the promoter to restore it to the structure close to a strong 70 promoterJust like this :Fig 16-15 cIn this new configuration , RNA polymerase can efficiently initiate transcription. Fig 16-15 Some repressors hold RNA polymerase a

35、t the promoter rather than excluding it Repressors work in different ways :By binding to a site overlapping the promoter, it blocks RNA polymerase binding. (lac repressor)The protein holds the promoter in a conformation incompatible with tanscription initiation.(the MerR case)Blocking the transition

36、 from the closed to open complex. Repressors bind to sites beside a promoter, interact with polymerase bound at that promoter and inhibit initiation. (E.coli Gal repressor)EXAMPLE FOUR-araBAD OPERON AraC and control of the araBAD operon by antiactivationThe promoter of the araBAD operon from E. coli

37、 is activated in the presence of arabinose (阿拉伯糖阿拉伯糖) and the absence of glucose and directs expression of genes encoding enzymes required for arabinose metabolism.Different from the Lac operon, two activators AraC and CAP work together to activate the araBAD operon expressionFig 16-18The magnitude

38、of induction of the araBAD promoter by arabinose is very large , and for this reason the promoter is often used in expression vectors.Expression vectors are DNA constructs in which efficient synthesis of any protein can be ensured by fusing a gene to a strong promoter . Part ThreePart ThreeCHAPTER 1

39、6 Gene Regulation in Prokaryotes Amino acid biosynthetic operons are controlled by premature transcription terminationthe tryptophan operon:Fig 16-19The trp operon encodes five structural genes required for tryptophan synthesis.These genes are regulated to efficiently express only when tryptophan is

40、 limiting.There are two layers of regulation involved: (1) transcription repression by the Trp repressor (initiation); (2) attenuationThe Trp repressor -the first layer of regulationWhen tryptophan is present, it binds the Trp repressor and induces a conformational change in that protein, enabling i

41、t to bind the trp operator and prevent transcription.When the tryptophan concentration is low, the Trp repressor is free of its corepressor and vacates its operator , allowing the synthesis of trp mRNA to commence from the adjacent promoter.The ligand that controls the activity of the trp repressor

42、acts not as an inducer but as a corepressor. Attenuation -the second layer of regulationThe key to understanding attenuation came from examining the suquence of the 5 end of trp operon mRNA.161 nucleotides of RNA are made from tryptophan promoter before RNA polymerase encounters the first codon of t

43、rpE.Near the end of this leader sequence ,and before trpE , is a transcription terminator, composed of a characteristic hairpin loop in the RNA.The hairpin loop is followed by 8 uridine residues. At this so-called attenuator , transcription usually stops,yielding a leader RNA 139 nucleotides long.Fi

44、g 16-20Thre features of the leader sequence: There is a second hairpin (besides the terminator hairpin) that can form between regions 1 and 2 of the leader sequence. region 2 also is complementary to region 3; thus , yet another hairpin consisting of regions 2 and 3 can form and when it does prevent

45、 the terminator hairpin (3,4) from forming.The leader RNA contains an open-reading frame encoding a short leader peptide of 14 amino acids, and this open-reading frame is preceded by a strong ribosome binding site.The sequence encoding the leader peptide has a striking feature : two tyrptophan codon

46、s in a row.The fuction of these codons is to stop a ribosome attempting to translate the leader peotide.Above all , how transcription termination at the trp operon attenuator is controlled by the availability of tryptophan?Fig 16-21The Importance of Attenuation nUse of both repression and attenuatio

47、n allows a fine tuning of the level of the intracellular tryptophan.nAttenuation alone can provide robust regulation: other amino acids operons like his and leu have no repressors and rely entirely on attenuation for their regulation.nProvides an example of regulation without the use of a regulatory

48、 protein, but using RNA structure instead.1.A typical negative feed-back regulation. Ribosomal Protein Are Translational Repressors of their Own SynthesisThe ribosome protein synthesis has challenges: Each ribosome contains some 50 distinct proteins that must be made at the same rate The rate of the

49、 ribosome protein synthesis is tightly closed to the cells growth rate How to overcome the challenges: Control of ribosome protein genes is simplified by their organization to several operons , each containing genes for up to 11 ribisomal proteins. Some nonribosomal proteins whose synthesis is also

50、linked to growth rate are contained in these operons, including those for RNAP subunits a, b and b. The primary control is at the level of translation, not transcription.How to overcome the challenges:nFor each operon,one ribosomal protein binds the messenger near the translation initiation sequence

51、 of the first genes in the operon, preventing ribosomes from binding and initiating translation.nRepressing translation of the first gene also prevents expression of some or all of the rest.nThe strategy is very sensitive. A few unused molecule of protein L4, for example, will shut down synthesis of

52、 that protein and other proteins in this operon. Ribosomal protein operonsThe protein that acts as a translational repressor of the other proteins is shaded red.Fig 16-22 The mechanism of one ribosomal protein also functions as a regulator of its own translation: the protein binds to the similar sit

53、es on the ribosomal RNA and to the regulated mRNAFig 16-23Part FourCHAPTER 16 Gene Regulation in ProkaryotesThe Case of Phage The Case of Phage : : Layers of RegulationLayers of RegulationBacteriophage is a virus that infects E.coli. Upon infection, the phage can propagate in either of two ways: lytically or lysogenically.A lysogen is extremely stable under normal circumstances ,but the phage dormant within it-the prophage-can efficiently switch to lytic growth if the cell is exposed to agents