分子生物學(xué)教學(xué)課件：CHAPTER 16 Gene Regulation in Prokaryotes

1、1 Chapter 16 Gene Regulation in Prokaryotes pPrinciples of Transcriptional Regulation pRegulation of Transcription Initiation: Examples from Bacteria:Lac operon alternative s factors, NtrC,MerR, Gal rep araBAD operon pExamples of Gene Regulation after Transcription Initiation pThe Case of Phage : La

2、yers of Regulation pTranscription initiation pElongation and termination: Antitermination and beyond Principles of transcriptional regulation Regulatory proteins activators: help RNA polymerase bind DNA after RNA polymerase binding Repressors: block that binding others Mechanism: Cooperative binding

3、 allostery Action at a distance and DNA looping Gene Expression is Controlled by Regulatory Proteins Gene expression is very often controlled by Extracellular Signals, which are communicated to genes by regulatory proteins : Positive regulators or activators increase the transcription Negative regul

4、ators or repressors decrease or eliminate the transcription a. Absence of Regulatory Proteins (operator) b. To Control Expression c. To Activate Expression Fig 16-1 Recruitment: cooperative binding of proteins to DNA Regulatory proteins activators: help RNA polymerase bind DNA after RNA polymerase b

5、inding Repressors: block that binding others Mechanism: Cooperative binding allostery Action at a distance and DNA looping Allostery Regulate Steps after RNA Polymerase Binding Targeting transition to the open complex Examples: Activator promoter NtrCglnA MerRmerT nSome promoters are inefficient at

6、more than one step and can be activated by more than one mechanism nRepressors can work in ways other than just blocking the promoter binding. For example, inhibition of the transition to the open complex. Regulatory proteins activators: help RNA polymerase bind DNA after RNA polymerase binding Repr

7、essors: block that binding others Mechanism: Cooperative binding allostery Action at a distance and DNA looping Action at a Distance and DNA Looping. Some proteins interact with each other even when bound to sites well separated on the DNA DNA-binding protein can facilitate interaction between DNA-b

8、inding proteins at a distance Targeting termination and beyond: Antitermination and Beyond The bulk of gene regulation takes place at the initiation of transcription. Some involve transcriptional elongation/termination, RNA processing, and translation of the mRNA into protein. pPrinciples of Transcr

9、iptional Regulation pRegulation of Transcription Initiation: Examples from Bacteria:Lac operon alternative s factors, NtrC,MerR, Gal rep araBAD operon pExamples of Gene Regulation after Transcription Initiation :(Trp operon) pThe Case of Phage : Layers of Regulation a unit of prokarytoic gene expres

10、sion and regulation which typically includes: 1. Structural genes for enzymes in a specific biosynthetic pathway whose expression is coordinately controlled. 2. Control elements, such as operator sequence. 3. Regulator gene(s) whose products recognize the control elements. Sometimes are encoded by t

11、he gene under the control of a different promoter Lactose operon Lactose operon 1.An activator and a repressor together control the lac genes The activator: CAP (Catabolite Activator Protein) or CRP (cAMP Receptor Protein); responses to the glucose level. The repressor: lac repressor that is encoded

12、 by LacI gene; responses to the lactose. CAP and lac repressor have opposing effects on RNA polymerase binding to the lac promoter The site bound by lac repressor is called the lac operator. The lac operator overlaps promoter, and so repressor bound to the operator physically prevents RNA polymerase

13、 from binding to the promoter. pCAP binds to a site with the similar structure as the operator, which is 60 bp upstream of the start site of transcription. pCAP also interacts with the enzyme and recruit it to the promoter. a CTD: C-terminal domain of the a subunit of RNAP CAP has separate activatin

14、g and DNA- binding surface CAP binds as a dimer a a CTD Fig 16-10 CAP and lac repressor bind DNA using a common structural motif nBoth CAP and lac repressor bind DNA using a helix-turn-helix motif. nOne is the recognition helix that can fits into the major groove of the DNA. DNA binding by a helix-t

15、urn-helix motif Fig 16-12 Hydrogen Bonds between l repressor and the major groove of the operator pLac repressor binds as a tetramer, with each operator is contacted by a repressor dimer. In addition to the primary operator, there are two other lac operators located 400 bp downstream and 90 bp upstr

16、eam, respectively. Not all the binding use a helix-turn-helix motif The activity of Lac repressor and CAP are controlled allosterically by their signals Binding of the corresponding signals alter the structure of these two regulatory proteins Polymerase binding to promoter is enhanced by active CAP

17、Very low transcriptionVery low transcription Low transcriptionLow transcription maximum transcriptionmaximum transcription pA regulator (CAP) works together with different repressor at different genes, this is an example of Combinatorial Control. pIn fact, CAP acts at more than 100 genes in E.coli,

18、working with an array of partners. 2.Combinatorial Control (組合調(diào)控組合調(diào)控): CAP controls other genes as well Regulatory proteins Activators: Repressors: Cooperative binding: allostery : block that binding others Lactose operon lac repressor The activity of Lac repressor and CAP CAP allostery Promoter rec

19、ognition : Alternative s factor direct RNA polymerase to alternative site of promoters Second example: Alternative s s factor Different 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 requirem

20、ents of transcription under normal and extreme growth condition E. coli : Heat shock s s32 Sporulation in Bacillus subtilis Bacteriophage factors Heat shock pAround 17 proteins are specifically expressed in E. coli when the temperature is increased above 37C. pThese proteins are expressed through tr

21、anscription by RNA polymerase using an alternative s factor s32 coded by rhoH gene. s32 has its own specific promoter consensus sequences. Many bacteriophages synthesize their own factors to endow the host RNA polymerase with a different promoter specificity and hence to selectively express their ow

22、n phage genes. Bacteriophages phage expresses a cascade of factors which allow a defined sequence of expression of different phage genes . Express early genes Encode 28 Encode factor for transcription of late genes Express middle genes Encode 34 NtrC and MerR: Transcriptional activators that work by

23、 allostery rather than by recruitment NtrC activator induces a conformational change in the enzyme, triggering transition to the open complex MerR activator causes the allosteric effect on the DNA and triggers the transition to the open complex NtrC has ATPase activity and works from DNA sites far f

24、rom the gene pNtrC controls expression of genes involved in nitrogen metabolism, such as the glnA gene pNtrC has separate activating and DNA- binding domains, and binds DNA only when the nitrogen levels are low. Low nitrogen levels NtrC phosphorylation and conformational change The DNA binding domai

25、n binds DNA sites at -150 position NtrC interacts with s54 (glnA promoter recognition) ATP hydrolysis and conformation change in polymerase transcription STARTs MerR activates transcription by twisting promoter DNA pMerR controls a gene called merT, which encodes an enzyme that makes cells resistant

26、 to the toxic effects of mercury pIn the presence of mercury, MerR binds to a sequence between 10 and 35 regions of the merT promoter and activates merT expression. As a s70 promoter, merT contains 19 bp between 10 and 35 elements (the typical length is 15-17 bp), leaving these two elements neither

27、optimally separated nor aligned. Structure of a merT-like promoter. moter with a 19bp spacer. b. promoter with a 19bp spacer when in complex with active activator. c. promoter with a 17bp spacer. Hg2+ -Hg2+ : -MerR binds to the promoter and locks it in the unfavorable conformation +Hg2+: MerR b

28、inds Hg2+ and undergo conformational change, which twists the promoter to restore it to the structure close to a strong s70 promoter nBlocking RNA polymerase binding through binding to a site overlapping the promoter. Lac repressor nBlocking the transition from the closed to open complex. Repressors

29、 bind to sites beside a promoter, interact with polymerase bound at that promoter and inhibit initiation. E.coli Gal repressor nLocking the promoter in a conformation incompatible with transcription initiation P4 protein from a bacteriophage Repressors work in many ways: Some repressors hold RNA pol

30、ymerase at the promoter rather than excluding it 條件條件 表達(dá)表達(dá) 有有Glu有 有Gal P2啟動(dòng)啟動(dòng)S2開始轉(zhuǎn)錄開始轉(zhuǎn)錄gal E， 組成型表達(dá)組成型表達(dá) 有有Glu無無Gal OE和 和OI 相互作用，成環(huán)，相互作用，成環(huán)， 轉(zhuǎn)錄只進(jìn)行轉(zhuǎn)錄只進(jìn)行20堿基便停止堿基便停止 無無Glu無無Gal P1不啟動(dòng)不啟動(dòng) 無無Glu有有GalP1啟動(dòng)啟動(dòng),3個(gè)基因轉(zhuǎn)錄個(gè)基因轉(zhuǎn)錄 n AraC and control of the araBAD operon by antiactivation nThe promoter of the araBAD o

31、peron 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. pDifferent from the Lac operon, two activators AraC and CAP work together to acti

32、vate the araBAD operon expression Function of AraC in regulating gene expression at the ara promoter pBAD Regions of contact RNAP a AraC dimer Regulatory proteins Activators: Repressors: Cooperative binding: allostery : block that binding Blocking the transition to open complex Locking the promoter

33、lac repressor The activity of Lac repressor and CAP CAP Gal repressor P4 protein from a bacteriophage Combinatorial Control AraC AraC pPrinciples of Transcriptional Regulation pRegulation of Transcription Initiation: Examples from Bacteria pExamples of Gene Regulation after Transcription Initiation

34、Trp operator Ribosomal proteins pThe Case of Phage : Layers of Regulation Amino acid biosynthetic operons are controlled by premature transcription termination: the tryptophan operon nThe trp operon encodes five structural genes required for tryptophan synthesis. nThese genes are regulated to effici

35、ently express only when tryptophan is limiting. nTwo layers of regulation are involved: (1) transcription repression by the Trp repressor (initiation); (2) attenuation Ribosomal proteins are translational repressors of their own synthesis Challenges the ribosome protein synthesis: 1.Each ribosome co

36、ntains some 50 distinct proteins that must be made at the same rate 2. The rate of the ribosome protein synthesis is tightly closed to the cells growth rate Ribosomal protein operons The protein that acts as a translational repressor of the other proteins is shaded red. The mechanism of one ribosoma

37、l protein also functions as a regulator of its own translation: the protein binds to the similar sites on the ribosomal RNA and to the regulated mRNA Fig 16-23 pPrinciples of Transcriptional Regulation pRegulation of Transcription Initiation: Examples from Bacteria:Lac operon alternative s factors,

38、NtrC,MerR, Gal rep araBAD operon pExamples of Gene Regulation after Transcription Initiation pThe Case of Phage : Layers of Regulation Bacteriophage is a virus that infects E.coli. Upon infection, the phage can propagate in either of two ways: lytically or lysogenically. p has a 50-kb genome and som

39、e 50 genes. Most of these encode coat proteins, proteins involved in DNA replication, recombination and lysis. Alternative patterns of gene expression control lytic and lysogenic growth Promoters in the right and left control regions of phage pThe depicted region contains two genes( cI and cro) and

40、three promoters. (PR, PL, PRM) Transcription in the control regions in lytic and lysogenic growth Regulatory proteins and their binding sites The cI encodesrepressor, a protein of two domains joined by a flexible linker region. Relative positions of promoter and operator sites in OR repressorCro Rep

41、ressor Binds to Operator Sites Cooperatively The repressor monomers interact to form dimers, and those dimers interact to form tetramers. These interaction ensure that binding of repressor to DNA is cooperative. The action of repressor and Cro pRepressor bound to OR1 and O R2 turns off transcription

42、 from PR. pRepressor bound at OR2 contacts RNA polymerase at PRM activating expression of the cI gene. pOR3 lies within PRM ;Cro bound there represses transcription of cI. Resspressor and Cro bind in Different Patterns to Control Lytic and Lysogenic Growth pFor lytic growth , a single Cro dimer is b

43、ound to OR3 ; this site overlaps PRM and so Cro represses that promoter. PR binds RNA polymerase and directs transcription of lytic genes; PL does likewise. pDuring lysogeny, PRM is on, while PR and PL are off.repressor bound cooperatively at OR1and OR2 blocks RNA polymerase binding at PR repressing

44、 transcription from that promoter. How the lysogenic state switches to the alternative lytic one Lysogenic induction requires proteolytic cleavage of repressor pE.coli senses and responds to DNA damage. It does this by activating the function of a protein called RecA. pActivated RecA stimulates auto

45、cleavage of LexA, releasing repression of those genes. This is called the SOS response. pIf the cell is a lysogen, repressor has evolved to resemble LexA, ensuring that repressor too undergoes autocleavage in response to activated RecA. The level of repressor in a lysogen must be tightly regulated.

46、pRepressor ensures its level never drops too low: it activates its own expression ,an example of positive autoregulation. pRepressor ensure its level never get too high, repressor also regulates itself negatively.( negative autoregulation ) Negative Autoregulation of Repressor Requires Long-Distance

47、 Interactions and a Large DNA Loop interaction of repressors at OR and OL Another Activator , c, Controls the Decision between Lytic and Lysogenic Growth upon Infection of a New Host. Determine which way the phage choose in the first place Genes and prompters involved in the lytic / lysogenic choice Growth Conditions of E.coli control the Stability of Cprotein and Lytic/Lysogenic Choice Establishment of lysogeny pCII: unstable protein pFstH: hfl gene encoding, protease regulated by growth condition of bacter