Chapter 10

1. Chapter 10 The operon

2. 10.1 Introduction 10.2 Regulation can be negative or positive 10.3 Structural gene clusters are coordinately controlled 10.4 The lac genes are controlled by a repressor 10.5 The lac operon can be induced 10.6 Repressor is controlled by a small molecule inducer 10.7 cis-acting constitutive mutations identify the operator 10.8 trans-acting mutations identify the regulator gene 10.9 Multimeric proteins have special genetic properties 10.10 Repressor protein binds to the operator 10.11 Binding of inducer releases repressor from the operator 10.12 Repressor is a tetramer 10.13 Repressor binds to three operators and interacts with RNA polymerase10.14 Repressor is always bound to DNA 10.15 The operator competes with low-affinity sites to bind repressor 10.16 Repression can occur at multiple loci

3. 10.17 Distinguishing positive and negative control 10.18 Catabolite repression involves the inducer cyclic AMP and the activator CAP10.19 CAP functions in different ways in different target operons 10.20 CAP bends DNA 10.21 The stringent response produces (p)ppGpp 10.22 (p)ppGpp is produced by the ribosome 10.23 pGpp has many effects 10.24 Translation can be regulated 10.25 r-protein synthesis is controlled by autogeneous regulation 10.26 Phage T4 p32 is controlled by an autogenous circuit 10.27 Autogenous regulation is often used to control synthesis of macromolecular assemblies 10.28 Alternative secondary structures control attenuation 10.29 The tryptophan operon is controlled by attenuation 10.30 Attenuation can be controlled by translation 10.31 Small RNA molecules can regulate translation 10.32 Antisense RNA can be used to inactivate gene expression

4. Operatoris the site on DNA at which a repressor protein binds to prevent transcription from initiating at the adjacent promoter.Repressor proteinbinds to operator on DNA or RNA to prevent transcription or translation, respectively.Structural genecodes for any RNA or protein product other than a regulator. 10.1 Introduction

5. Figure 10.1 A regulator gene codes for a protein that acts at a target site on DNA. 10.1 Introduction

6. 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 coordinately. 10.1 Introduction

7. 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. 10.1 Introduction

8. 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 Structural gene clusters are coordinately controlled

9. 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 the lacY and lacA genes and a terminator. 10.2 Structural gene clusters are coordinately controlled

10. 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

11. Allosteric control refers to the ability 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 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 present; 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 transcription (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

12. 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 is followed by rapid cessation of synthesis. 10.3 Repressor is controlled by a small molecule inducer

13. 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 transcription. 10.3 Repressor is controlled by a small molecule inducer

14. 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 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

15. 10.4 Mutations identify the operator and the regulator gene Figure 10.8Operator 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.

16. 10.4 Mutations identify the operator and 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.

17. 10.4 Mutations identify the operator and the regulator gene Figure 10.10 Mutations map the regions of the lacl gene 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 between residues 62-300.

18. 10.5 Repressor protein binds to the operator and is released by inducer 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.

19. 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

20. 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 to repressor bound at the operator (right)?

21. 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.

22. 10.5 Repressor protein binds to the operator 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.

23. 10.5 Repressor protein binds to the operator 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.

24. 10.5 Repressor protein binds to the operator 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.

25. 10.5 Repressor protein binds to the operator 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.

26. 10.5 Repressor protein binds to the operator 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.

27. 10.5 Repressor protein binds to the operator and is released by 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.

28. 10.6 The specificity of protein-DNA interactions Figure 10.17 Lac repressor binds strongly and specifically to its operator, but is released by inducer. All equilibrium constants are in M-1.

29. 10.6 The specificity of protein-DNA interactions Figure 10.18 Virtually all the repressor in the cell is bound to DNA.

30. 10.6 The specificity of protein-DNA interactions Figure 9.12How does RNA polymerase find target promoters so rapidly on DNA?

31. Autogenous control describes the action of a gene product that either inhibits (negative autogenous control) or activates (positive autogenous control) expression of the gene coding for it. 10.7 Repression can occur at multiple loci

32. 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 arrows.

33. 10.7 Repression can occur at multiple loci Figure 10.20 Operators may lie at various positions relative to the promoter.

34. Derepressed state describes a gene that is turned on. It is synonymous with induced when describing the normal state of a gene; it has the same meaning as constitutive in describing the effect of mutation. 10.8 Distinguishing positive and negative control

35. 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 coordinately.

36. 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.

37. 10.8 Distinguishing 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.

38. 10.9 Catabolite repression involves positive regulation at the promoter Catabolite repression describes the decreased expression of many bacterial operons that results from addition of glucose. It is caused by a decrease in the level of cyclic AMP, which in turn inactivates the CAP regulator.

39. 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 the sugar ring.

40. 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.

41. 10.9 Catabolite repression involves positive regulation at the promoter Figure 10.23 Glucose causes catabolite repression by reducing the level of cyclic AMP.

42. 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).

43. 10.9 Catabolite repression involves positive regulation at the promoter Figure 10.25 The CAP protein can bind at different sites relative to RNA polymerase.

44. 10.9 Catabolite repression involves positive regulation at the promoter Figure 10.26 Gel electrophoresis can be used to analyze bending.

45. 10.9 Catabolite repression involves positive regulation at the promoter Figure 10.27 CAP bends DNA >90° around the center of symmetry.

46. 10.10 Adverse growth conditions provoke the stringent response Idling reaction is the production of pppGpp and ppGpp by ribosomes when an uncharged tRNA is present in the A 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.

47. 10.10 Adverse growth conditions provoke the stringent response Figure 10.28 Stringent factor catalyzes the synthesis of pppGpp and ppGpp; ribosomal proteins can dephosphorylate pppGpp to ppGpp.

48. 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 movement 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.

49. 10.10 Adverse growth conditions provoke the stringent response Figure 10.30 Nucleotide levels control initiation of rRNA transcription.

50. 10.10 Adverse 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.