分子生物学 第十章 操纵子（The operon）

1、Chapter 10,The operon,10.1 Introduction10.2 Regulation can be negative or positive10.3 Structural gene clusters are coordinately controlled10.4 The lac genes are controlled by a repressor10.5 The lac operon can be induced10.6 Repressor is controlled by a small molecule inducer 10.7 cis-acting consti

2、tutive mutations identify the operator10.8 trans-acting mutations identify the regulator gene10.9 Multimeric proteins have special genetic properties10.10 Repressor protein binds to the operator10.11 Binding of inducer releases repressor from the operator10.12 Repressor is a tetramer10.13 Repressor

3、binds to three operators and interacts with RNA polymerase10.14 Repressor is always bound to DNA10.15 The operator competes with low-affinity sites to bind repressor10.16 Repression can occur at multiple loci,10.17 Distinguishing positive and negative control 10.18 Catabolite repression involves the

4、 inducer cyclic AMP and the activator CAP10.19 CAP functions in different ways in different target operons10.20 CAP bends DNA10.21 The stringent response produces (p)ppGpp10.22 (p)ppGpp is produced by the ribosome10.23 pGpp has many effects10.24 Translation can be regulated10.25 r-protein synthesis

5、is controlled by autogeneous regulation10.26 Phage T4 p32 is controlled by an autogenous circuit10.27 Autogenous regulation is often used to control synthesis of macromolecular assemblies10.28 Alternative secondary structures control attenuation10.29 The tryptophan operon is controlled by attenuatio

6、n10.30 Attenuation can be controlled by translation10.31 Small RNA molecules can regulate translation10.32 Antisense RNA can be used to inactivate gene expression,Operator is the site on DNA at which a repressor protein binds to prevent transcription from initiating at the adjacent promoter.Represso

7、r protein binds to operator on DNA or RNA to prevent transcription or translation, respectively.Structural gene codes for any RNA or protein product other than a regulator.,10.1 Introduction,Figure 10.1 A regulator gene codes for a protein that acts at a target site on DNA.,10.1 Introduction,Figure

8、10.2 In negative control, a trans-acting repressor binds to the cis-acting operator to turn off transcription. In prokaryotes, multiple genes are controlled coordinately.,10.1 Introduction,Figure 10.3 In positive control, trans-acting factors must bind to cis-acting sites in order for RNA polymerase

9、 to initiate transcription at the promoter. In a eukaryotic system, a structural gene is controlled individually.,10.1 Introduction,Operon is a unit of bacterial gene expression and regulation, including structural genes and control elements in DNA recognized by regulator gene product(s).,10.2 Struc

10、tural gene clusters are coordinately controlled,Figure 10.4 The lac operon occupies 6000 bp of DNA. At the left the lacI gene has its own promoter and terminator. The end of the lacI region is adjacent to the promoter, P. The operator, O, occupies the first 26 bp of the long lacZ gene, followed by t

11、he lacY and lacA genes and a terminator.,10.2 Structural gene clusters are coordinately controlled,Figure 10.5 Repressor and RNA polymerase bind at sites that overlap around the startpoint of the lac operon.,10.2 Structural gene clusters are coordinately controlled,Allosteric control refers to the a

12、bility of an interaction at one site of a protein to influence the activity of another site.Coordinate regulation refers to the common control of a group of genes.Corepressor is a small molecule that triggers repression of transcription by binding to a regulator protein.Gratuitous inducers resemble

13、authentic inducers of transcription but are not substrates for the induced enzymes.Inducer is a small molecule that triggers gene transcription by binding to a regulator protein.Induction refers to the ability of bacteria (or yeast) to synthesize certain enzymes only when their substrates are presen

14、t; applied to gene expression, refers to switching on transcription as a result of interaction of the inducer with the regulator protein.Repression is the ability of bacteria to prevent synthesis of certain enzymes when their products are present; more generally, refers to inhibition of transcriptio

15、n (or translation) by binding of repressor protein to a specific site on DNA (or mRNA).,10.3 Repressor is controlled by a small molecule inducer,Figure 10.6 Addition of inducer results in rapid induction of lac mRNA, and is followed after a short lag by synthesis of the enzymes; removal of inducer i

16、s followed by rapid cessation of synthesis.,10.3 Repressor is controlled by a small molecule inducer,Figure 10.7 Repressor maintains the lac operon in the inactive condition by binding to the operator; addition of inducer releases the repressor, and thereby allows RNA polymerase to initiate transcri

17、ption.,10.3 Repressor is controlled by a small molecule inducer,Interallelic complementation describes the change in the properties of a heteromultimeric protein brought about by the interaction of subunits coded by two different mutant alleles; the mixed protein may be more or less active than the

18、protein consisting of subunits only of one or the other type.Negative complementation occurs when interallelic complementation allows a mutant subunit to suppress the activity of a wild-type subunit in a multimeric protein.,10.4 Mutations identify the operator and the regulator gene,Figure 10.8Opera

19、tor mutations are constitutive because the operator is unable to bind repressor protein; this allows RNA polymerase to have unrestrained access to the promoter. The Oc mutations are cis-acting, because they affect only the contiguous set of structural genes.,10.4 Mutations identify the operator and

20、the regulator gene,Figure 10.9 Mutations that inactivate the lacI gene cause the operon to be constitutively expressed, because the mutant repressor protein cannot bind to the operator.,10.4 Mutations identify the operator and the regulator gene,Figure 10.10 Mutations map the regions of the lacl gen

21、e responsible for different functions. The DNA-binding domain is identified by lacI-d mutations at the N-terminal region; lacl- mutations unable to form tetramers are located between residues 220-280. Other lacI- mutations occur throughout the gene. lacIs mutations occur in regularly spaced clusters

22、 between residues 62-300.,10.4 Mutations identify the operator and the regulator gene,Figure 10.11 The lac operator has a symmetrical sequence. The sequence is numbered relative to the startpoint for transcription at +1. The regions of dyad symmetry are indicated by the shaded blocks.,10.5 Repressor

23、 protein binds to the operator and is released by inducer,Figure 9.16 One face of the promoter contains the contact points for RNA.,10.5 Repressor protein binds to the operator and is released by inducer,Figure 10.12 Does the inducer bind to free repressor to upset an equilibrium (left) or directly

24、to repressor bound at the operator (right)?,10.5 Repressor protein binds to the operator and is released by inducer,Figure 10.13 The structure of a monomer of Lac repressor identifies several independent domains. Photograph kindly provided by Mitchell Lewis.,10.5 Repressor protein binds to the opera

25、tor and is released by inducer,Figure 10.14 The crystal structure of the core region of Lac repressor identifies the interactions between monomers in the tetramer. Each monomer is identified by a different color. Photographs kindly provided by Alan Friedman.,10.5 Repressor protein binds to the opera

26、tor and is released by inducer,Figure 10.14 The crystal structure of the core region of Lac repressor identifies the interactions between monomers in the tetramer. Each monomer is identified by a different color. Photographs kindly provided by Alan Friedman.,10.5 Repressor protein binds to the opera

27、tor and is released by inducer,Figure 10.14 The crystal structure of the core region of Lac repressor identifies the interactions between monomers in the tetramer. Each monomer is identified by a different color. Photographs kindly provided by Alan Friedman.,10.5 Repressor protein binds to the opera

28、tor and is released by inducer,Figure 10.14 The crystal structure of the core region of Lac repressor identifies the interactions between monomers in the tetramer. Each monomer is identified by a different color. Photographs kindly provided by Alan Friedman.,10.5 Repressor protein binds to the opera

29、tor and is released by inducer,Figure 10.15 Inducer changes the structure of the core so that the headpieces of a repressor dimer are no longer in an orientation that permits binding to DNA. Photographs kindly provided by Mitchell Lewis.,10.5 Repressor protein binds to the operator and is released b

30、y inducer,Figure 10.16 When a repressor tetramer binds to two operators, the stretch of DNA between them is forced into a tight loop. (The blue structure in the center of the looped DNA represents CAP, another regulator protein that binds in this region). Photograph kindly provided by Mitchell Lewis

31、.,10.5 Repressor protein binds to the operator and is released by inducer,Figure 10.17 Lac repressor binds strongly and specifically to its operator, but is released by inducer. All equilibrium constants are in M-1.,10.6 The specificity of protein-DNA interactions,Figure 10.18 Virtually all the repr

32、essor in the cell is bound to DNA.,10.6 The specificity of protein-DNA interactions,Figure 9.12How does RNA polymerase find target promoters so rapidly on DNA?,10.6 The specificity of protein-DNA interactions,Autogenous control describes the action of a gene product that either inhibits (negative au

33、togenous control) or activates (positive autogenous control) expression of the gene coding for it.,10.7 Repression can occur at multiple loci,Figure 10.19 The trp repressor recognizes operators at three loci. Conserved bases are shown in red. The location of the mRNA varies, as indicated by the red

34、arrows.,10.7 Repression can occur at multiple loci,Figure 10.20 Operators may lie at various positions relative to the promoter.,10.7 Repression can occur at multiple loci,Derepressed state describes a gene that is turned on. It is synonymous with induced when describing the normal state of a gene;

35、it has the same meaning as constitutive in describing the effect of mutation.,10.8 Distinguishing positive and negative control,Figure 10.2 In negative control, a trans-acting repressor binds to the cis-acting operator to turn off transcription. In prokaryotes, multiple genes are controlled coordina

36、tely.,10.8 Distinguishing positive and negative control,Figure 10.3 In positive control, trans-acting factors must bind to cis-acting sites in order for RNA polymerase to initiate transcription at the promoter. In a eukaryotic system, a structural gene is controlled individually.,10.8 Distinguishing

37、 positive and negative control,Figure 10.21 Control circuits are versatile and can be designed to allow positive or negative control of induction or repression.,10.8 Distinguishing positive and negative control,Catabolite repression describes the decreased expression of many bacterial operons that r

38、esults from addition of glucose. It is caused by a decrease in the level of cyclic AMP, which in turn inactivates the CAP regulator.,10.9 Catabolite repression involves positive regulation at the promoter,Figure 10.22 Cyclic AMP has a single phosphate group connected to both the 3 and 5 positions of

39、 the sugar ring.,10.9 Catabolite repression involves positive regulation at the promoter,Figure 10.21 Control circuits are versatile and can be designed to allow positive or negative control of induction or repression.,10.9 Catabolite repression involves positive regulation at the promoter,Figure 10

40、.23 Glucose causes catabolite repression by reducing the level of cyclic AMP.,10.9 Catabolite repression involves positive regulation at the promoter,Figure 10.24 The consensus sequence for CAP contains the well conserved pentamer TGTGA and (sometimes) an inversion of this sequence (TCANA).,10.9 Cat

41、abolite repression involves positive regulation at the promoter,Figure 10.25 The CAP protein can bind at different sites relative to RNA polymerase.,10.9 Catabolite repression involves positive regulation at the promoter,Figure 10.26 Gel electrophoresis can be used to analyze bending.,10.9 Catabolit

42、e repression involves positive regulation at the promoter,Figure 10.27 CAP bends DNA 90 around the center of symmetry.,10.9 Catabolite repression involves positive regulation at the promoter,Idling reaction is the production of pppGpp and ppGpp by ribosomes when an uncharged tRNA is present in the A

43、 site; triggers the stringent response.Stringent response refers to the ability of a bacterium to shut down synthesis of tRNA and ribosomes in a poor-growth medium.,10.10 Adverse growth conditions provoke the stringent response,Figure 10.28 Stringent factor catalyzes the synthesis of pppGpp and ppGp

44、p; ribosomal proteins can dephosphorylate pppGpp to ppGpp.,10.10 Adverse growth conditions provoke the stringent response,Figure 10.29 In normal protein synthesis, the presence of aminoacyl-tRNA in the A site is a signal for peptidyl transferase to transfer the polypeptide chain, followed by movemen

45、t catalyzed by EF-G; but under stringent conditions, the presence of uncharged tRNA causes RelA protein to synthesize (p)ppGpp and to expel the tRNA.,10.10 Adverse growth conditions provoke the stringent response,Figure 10.30 Nucleotide levels control initiation of rRNA transcription.,10.10 Adverse

46、growth conditions provoke the stringent response,Figure 10.35 Translation of the r-protein operons is autogenously controlled and responds to the level of rRNA.,10.10 Adverse growth conditions provoke the stringent response,Figure 10.31 A regulator protein may block translation by binding to a site

47、on mRNA that overlaps the ribosome-binding site at the initiation codon.,10.11 Autogenous control may occur at translation,Figure 10.32 Proteins that bind to sequences within the initiation regions of mRNAs may function as translational repressors.,10.11 Autogenous control may occur at translation,F

48、igure 10.33 Secondary structure can control initiation. Only one initiation site is available in the RNA phage, but translation of the first cistron changes the conformation of the RNA so that other initiation site(s) become available.,10.11 Autogenous control may occur at translation,Figure 10.34 G

49、enes for ribosomal proteins, protein synthesis factors, and RNA polymerase subunits are interspersed in a small number of operons that are autonomously regulated. The regulator is named in red; the proteins that are regulated are shaded in pink.,10.11 Autogenous control may occur at translation,Figu

50、re 10.35 Translation of the r-protein operons is autogenously controlled and responds to the level of rRNA.,10.11 Autogenous control may occur at translation,Figure 10.36 Excess gene 32 protein (p32) binds to its own mRNA to prevent ribosomes from initiating translation.,10.11 Autogenous control may

51、 occur at translation,Figure 10.37 Gene 32 protein binds to various substrates with different affinities, in the order single-stranded DNA, its own mRNA, and other mRNAs. Binding to its own mRNA prevents the level of p32 from rising 10-6 M.,10.11 Autogenous control may occur at translation,Figure 10

52、.38 Tubulin is assembled into microtubules when it is synthesized. Accumulation of excess free tubulin induces instability in the tubulin mRNA by acting at a site at the start of the reading frame in mRNA or at the corresponding position in the nascent protein.,10.11 Autogenous control may occur at

53、translation,Figure 10.39 Attenuation occurs when a terminator hairpin in RNA is prevented from forming.,10.12 Alternative secondary structures control attenuation,Figure 10.40 Termination can be controlled via changes in RNA secondary structure that are determined by ribosome movement.,10.13 Attenua

54、tion can be controlled by translation,Figure 10.41 The trp operon consists of five contiguous structural genes preceded by a control region that includes a promoter, operator, leader peptide coding region, and attenuator.,10.13 Attenuation can be controlled by translation,Figure 10.42 An attenuator

55、controls the progression of RNA polymerase into the trp genes. RNA polymerase initiates at the promoter and then proceeds to position 90, where it pauses before proceeding to the attenuator at position 140. In the absence of tryptophan, the polymerase continues into the structural genes (trpE starts

56、 at +163). In the presence of tryptophan there is 90% probability of termination to release the 140-base leader RNA.,10.13 Attenuation can be controlled by translation,Figure 10.43 The trp leader region can exist in alternative base-paired conformations. The center shows the four regions that can ba

57、se pair. Region 1 is complementary to region 2, which is complementary to region 3, which is complementary to region 4. On the left is the conformation produced when region 1 pairs with region 2, and region 3 pairs with region 4. On the right is the conformation when region 2 pairs with region 3, leaving regions 1 and 4 unpaired.,10.13 Attenuation can be controlled by translation,Figure 10.44 The alternatives for RNA polymerase at the attenuator depend on the location of the ribosome, which determines whether regions 3 and 4 can pair to form the