1. PowerPoint Presentation Materials to accompany Genetics: Analysis and Principles Robert J. Brooker Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display CHAPTER 14 GENE REGULATION IN BACTERIA AND BACTERIOPHAGES

2. INTRODUCTION The term gene regulation means that the level of gene expression can vary under different conditions Genes that are unregulated are termed constitutive They have essentially constant levels of expression Frequently, constitutive genes encode proteins that are necessary for the survival of the organism The benefit of regulating genes is that encoded proteins will be produced only when required 14-2 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

3. INTRODUCTION Gene regulation is important for cellular processes such as 1. Metabolism 2. Response to environmental stress 3. Cell division Regulation can occur at any of the points on the pathway to gene expression Refer to Figure 14.1 14-3 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

4. 14-4 Figure 14.1

5. The most common way to regulate gene expression in bacteria is at the transcriptional level The rate of RNA synthesis can be increased or decreased Negative control: Repressors  Bind to DNA and inhibit transcription Positive control: Activators  Bind to DNA and increase transcription Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 14.1 TRANSCRIPTIONAL REGULATION 14-5

6. In some cases, the presence of a small effector molecule may increase transcription These molecules are termed inducers Bind activators and cause them to bind to DNA Bind repressors and prevent them from binding to DNA In other cases, the presence of a small effector molecule may inhibit transcription Corepressors bind to repressors and cause them to bind to DNA Inhibitors bind to activators and prevent them from binding to DNA Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 14-6

7. Fig. 14.2ab(TE Art) Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Repressor protein, inducer molecule, inducible gene Repressor protein, corepressor molecule, repressible gene Promoter DNA Repressor protein No transcription Transcription occurs RNA polymerase Inducer Repressor protein Corepressor RNA polymerase or Transcription occurs Repressor protein No transcription or

8. Fig. 14.2cd(TE Art) Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Activator protein, inducer molecule, inducible gene Activator protein, inhibitor molecule, repressible gene No transcription Transcription occurs RNA polymerase Activator protein Inhibitor Inducer Activator protein Activator protein or Transcription occurs RNA polymerase Activator protein No transcription or

9. Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display At the turn of the 20 th century, scientists made the following observation A particular enzyme appears in the cell only after the cell has been exposed to the enzyme’s substrate This observation became known as enzyme adaptation François Jacob and Jacques Monod at the Pasteur Institute in Paris were interested in this phenomenon They focused their attention on lactose metabolism in E. coli to investigate this problem The Phenomenon of Enzyme Adaptation 14-9

10. Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display An operon is a regulatory unit consisting of a few structural genes under the control of one promoter It encodes polycistronic mRNA that contains the coding sequence for two or more structural genes This allows a bacterium to coordinately regulate a group of genes that encode proteins with a common function The lac Operon 14-10

11. Figure 16-3 Copyright © 2006 Pearson Prentice Hall, Inc.

12. 14-11 An operon contains several different regions There are two distinct transcriptional units 1. The actual lac operon a. DNA elements Promoter  Binds RNA polymerase Operator  Binds the lac repressor protein CAP site  Binds the Catabolite Activator Protein (CAP) b. Structural genes lacZ  Encodes  -galactosidase Enzymatically cleaves lactose and lactose analogues Also converts lactose into allolactose (an isomer) lacY  Encodes lactose permease Membrane protein required for transport of lactose and analogues lacA  Encodes transacetylase Covalently modifies lactose and analogues Its functional necessity remains unclear Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

13. 14-13 Figure 14.3 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

14. 14-12 Figure 14.3a shows the organization and transcriptional regulation of the lac operon genes There are two distinct transcriptional units Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 2. The lacI gene Not considered part of the lac operon Has its own promoter, the i promoter Constitutively expressed at fairly low levels Encodes the lac repressor The lac repressor protein functions as a tetramer Only a small amount of protein is needed to repress the lac operon

15. Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display The lac operon can be transcriptionally regulated 1. By a repressor protein 2. By an activator protein The first method is an inducible, negative control mechanism It involves the lac repressor protein The inducer is allolactose It binds to the lac repressor and inactivates it Refer to Figure 14.4 The lac Operon Is Regulated By a Repressor Protein 14-14

16. 14-15 Figure 14.4 Therefore no allolactose Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display Constitutive expression RNA pol cannot access the promoter The lac operon is now repressed

17. 14-16 Figure 14.4 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display The conformation of the repressor is now altered Some gets converted to allolactose Repressor can no longer bind to operator Translation The lac operon is now induced

18. Figure 16-5 Copyright © 2006 Pearson Prentice Hall, Inc.

19. 14-17 The cycle of lac operon induction and repression Figure 14.5 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display Repressor does not completely inhibit transcription So very small amounts of the enzymes are made

20. In the 1950s, Jacob and Monod, and their colleague Arthur Pardee, had identified a few rare mutant strains of bacteria with abnormal lactose adaptation One type of mutant involved a defect in the lacI gene It was designated lacI – It resulted in the constitutive expression of the lac operon even in the absence of lactose The lacI – mutations mapped very close to the lac operon Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display Experiment 14A: The lacI Gene Encodes a Repressor Protein 14-18

21. Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 14-19 Jacob, Monod and Pardee proposed two different functions for the lacI gene Figure 14.6 Jacob, Monod and Pardee applied a genetic approach to distinguish between the two hypotheses

22. Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 14-20 They used bacterial conjugation methods to introduce different portions of the lac operon into different strains They identified F’ factors (plasmids) that carried portions of the lac operon For example: Consider an F’ factor that carries the lacI gene Bacteria that receive this will have two copies of the lacI gene One on the chromosome and the other on the F’ factor These are called merozygotes, or partial diploids

23. Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 14-21 Merozygotes were instrumental in allowing Jacob et al to elucidate the function of the lacI gene There are two key points 1. The two lacI genes in a merozygote may be different alleles lacI – on the chromosome lacI + on the F’ factor 2. Genes on the F’ factor are not physically connected to those on the bacterial chromosome If hypothesis 1 is correct The repressor protein produced from the F’ factor can diffuse and regulate the lac operon on the bacterial chromosome If hypothesis 2 is correct The binding site on the F’ factor cannot affect the lac operon on the bacterial chromosome, because they are not physically adjacent

24. The Hypothesis The lacI gene either/or 1. Encodes a regulatory protein (the lac repressor) that can diffuse throughout the cell 2. Acts as a binding site for a repressor protein Thus it can only act on a physically connected lac operon Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display Testing the Hypothesis Refer to Figure 14.7 14-22

25. 14-23 Figure 14.7

26. 14-24

27. Figure 14.7 14-25

28. The Data 14-26 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display StrainAddition of lactose Amount of  -galactosidase (percentage of parent strain) MutantNo100% MutantYes100% MerozygoteNo<1% MerozygoteYes220%

29. Interpreting the Data 14-27 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display StrainAddition of lactose Amount of  -galactosidase (percentage of parent strain) MutantNo100% MutantYes100% MerozygoteNo<1% MerozygoteYes220% Expected result because of constitutive expression in the lacI – strain In the absence of lactose, both lac operons are repressed In the presence of lactose, both lac operons are induced, yielding a higher level of enzyme activity This result is consistent with hypothesis 1 The lacI gene codes for a repressor protein that can diffuse throughout the cell and bind to any lac operon

30. Interpreting the Data 14-28 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display The interaction between regulatory proteins and DNA sequences have led to two definitions 1. Trans-effect Genetic regulation that can occur even though DNA segments are not physically adjacent Mediated by genes that encode regulatory proteins Example: The action of the lac repressor on the lac operon 2. Cis-effect or cis-acting element A DNA sequence that must be adjacent to the gene(s) it regulates Mediated by sequences that bind regulatory proteins Example: The lac operator

31. Interpreting the Data 14-29 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display Table 14.1 summarizes the effects of lacI gene mutations versus lacO (operator) in merozygotes Overall A mutation in a trans-acting factor is complemented by the introduction of a second gene with a normal function However, a mutation in a cis-acting element is not!

32. Figure 16-6 Copyright © 2006 Pearson Prentice Hall, Inc.

33. 14-30 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

34. Figure 16-7 Copyright © 2006 Pearson Prentice Hall, Inc.

35. Table 16-1 Copyright © 2006 Pearson Prentice Hall, Inc.

36. Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display The lac operon can be transcriptionally regulated in a second way, known as catabolite repression When exposed to both lactose and glucose E. coli uses glucose first, and catabolite repression prevents the use of lactose When glucose is depleted, catabolite repression is alleviated, and the lac operon is expressed The lac Operon Is Also Regulated By an Activator Protein 14-31

37. Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display The small effector molecule in catabolite repression is not glucose This form of genetic regulation involves a small molecule, cyclic AMP (cAMP) It is produced from ATP via the enzyme adenylyl cyclase cAMP binds an activator protein known as the Catabolite Activator Protein (CAP) Also termed the cyclic AMP receptor protein (CRP) The lac Operon Is Also Regulated By an Activator Protein 14-32

38. Figure 16-9 Copyright © 2006 Pearson Prentice Hall, Inc.

39. Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display The cAMP-CAP complex is an example of genetic regulation that is inducible and under positive control The cAMP-CAP complex binds to the CAP site near the lac promoter and increases transcription In the presence of glucose, the enzyme adenylyl cyclase is inhibited This decreases the levels of cAMP in the cell Therefore, cAMP is no longer available to bind CAP The lac Operon Is Also Regulated By an Activator Protein 14-33

41. Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings Figure 18.23 Positive control of the lac operon by catabolite activator protein (CAP) Lactose present, glucose scarce (cAMP level high): abundant lac mRNA synthesized. If glucose is scarce, the high level of cAMP activates CAP, and the lac operon produces large amounts of mRNA for the lactose pathway. (a) CAP-binding site Operator Promoter RNA polymerase can bind and transcribe Inactive CAP Active CAP cAMP DNA Inactive lac repressor lacl lacZ

42. Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings (b) Lactose present, glucose present (cAMP level low): little lac mRNA synthesized. When glucose is present, cAMP is scarce, and CAP is unable to stimulate transcription. Inactive lac repressor Inactive CAP DNA RNA polymerase can’t bind Operator lacl lacZ CAP-binding site Promoter

43. Both negative and positive control mechanisms are used by the cell.

44. Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display Detailed genetic and crystallographic studies have shown that the binding of the lac repressor is more complex than originally thought In all, three operator sites have been discovered O 1  Next to the promoter O 2  Downstream in the lacZ coding region O 3  Slightly upstream of the CAP site Refer to Figure 14.9 The lac Operon Has Three Operator Sites for the lac Repressor 14-36

45. 14-37 The identification of three lac operator sites Figure 14.9 Repression is 1,300 fold Therefore, transcription is 1/1,300 the level when lactose is present No repression ie: Constitutive expression

46. Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display The results of Figure 14.9 supported the hypothesis that the lac repressor must bind to two of the three operators to cause repression It can bind to O 1 and O 2, or to O 1 and O 3 But not O 2 and O 3 If either O 2 or O 3 is missing maximal repression is not achieved Binding of the lac repressor to two operator sites requires that the DNA form a loop A loop in the DNA brings the operator sites closer together This facilitates the binding of the repressor protein 14-38

47. 14-39 Figure 14.10 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display Each repressor dimer binds to one operator site

48. Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display Another operon in E. coli that is involved in sugar metabolism is the ara (arabinose) operon It contains Three structural genes involved in arabinose metabolism These are designated araB, araA and araD A single promoter, P BAD A CAP site, which binds the catabolite activator protein The ara Operon 14-40

49. 14-41 Figure 14.11 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display The araC gene is adjacent to the ara operon It has its own promoter, P C It encodes a regulatory protein, AraC AraC can bind to three different operator sites Designated araI, araO 1 and araO 2 The AraC protein can act as either a negative or positive regulator of transcription Depending on whether or not arabinose is present

50. AraC protein binds to all three operators AraC dimer bound to araO 1 inhibits transcription of the araC gene This keeps AraC protein levels fairly low AraC monomers bound to araO 2 and araI repress the ara operon They bind to each other (via looped DNA), and block RNA pol access to P BAD araO 1 Figure 14.12 14-42

51. Arabinose binds to the AraC proteins The interaction betweem the AraC proteins at the araO 2 and araI is broken This breaks the DNA loop Another, AraC protein binds to araI This AraC dimer at araI activates transcription 14-43 CAP-cAMP activation occurs when glucose levels are low Figure 14.12

52. Figure 16-15 Copyright © 2006 Pearson Prentice Hall, Inc.

53. Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings Regulation of a anabolic pathway (a) Regulation of enzyme activity Enzyme 1 Enzyme 2 Enzyme 3 Enzyme 4 Enzyme 5 Regulation of gene expression Feedback inhibition Tryptophan Precursor (b) Regulation of enzyme production Gene 2 Gene 1 Gene 3 Gene 4 Gene 5 – –

54. Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display The trp operon (pronounced “trip”) is involved in the biosynthesis of the amino acid tryptophan The genes trpE, trpD, trpC, trpB and trpA encode enzymes involved in tryptophan biosynthesis The genes trpR and trpL are involved in regulation trpR  Encodes the trp repressor protein Functions in repression trpL  Encodes a short peptide called the Leader peptide Functions in attenuation The trp Operon 14-44

55. 14-45 Organization of the trp operon and regulation via the trp repressor protein Figure 14.13 Cannot bind to the operator site RNA pol can bind to the promoter

56. 14-46 Organization of the trp operon and regulation via the trp repressor protein Figure 14.13

58. 14-47 Organization of the trp operon and regulation via the trp repressor protein Figure 14.13 Another mechanism of regulation

59. Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display Attenuation occurs in bacteria because of the coupling of transcription and translation During attenuation, transcription actually begins but it is terminated before the entire mRNA is made A segment of DNA, termed the attenuator, is important in facilitating this termination In the case of the trp operon, transcription terminates shortly past the trpL region (Figure 14.13c) Thus attenuation inhibits the further production of tryptophan The segment of trp operon immediately downstream from the operator site plays a critical role in attenuation The first gene in the trp operon is trpL It encodes a short peptide termed the Leader peptide Refer to Figure 14.14 14-48

60. 14-49 Sequence of the trpL mRNA produced during attenuation Figure 14.14 These two codons provide a way to sense if there is sufficient tryptophan for translation The 3-4 stem loop is followed by a sequence of Uracils Region 2 is complementary to regions 1 and 3 Region 3 is complementary to regions 2 and 4 Therefore several stem-loops structures are possible It acts as an intrinsic terminator

61. Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display Formation of the 3-4 stem-loop causes RNA pol to terminate transcription at the end of the trpL gene Conditions that favor the formation of the 3-4 stem-loop rely on the translation of the trpL mRNA There are three possible scenarios 1. No translation 2. Low levels of tryptophan 3. High levels of tryptophan Refer to Figure 14.15 14-50

62. 14-51 Possible stem-loop structures formed from trpL mRNA under different conditions of translation Figure 14.15 Therefore no coupling of transcription and translation Most stable form of mRNA occurs Transcription terminates just past the trpL gene

63. 14-52 Possible stem-loop structures formed from trpL mRNA under different conditions of translation Figure 14.15 Insufficient amounts of tRNA trp Region 1 is blocked 3-4 stem-loop does not form RNA pol transcribes rest of operon

64. 14-53 Possible stem-loop structures formed from trpL mRNA under different conditions of translation Figure 14.15 Sufficient amounts of tRNA trp Translation of the trpL mRNA progresses until stop codon Region 2 cannot base pair with any other region 3-4 stem-loop forms Transcription terminates RNA polymerase pauses

65. Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display The study of many operons revealed a general trend concerning inducible versus repressible regulation Operons involved in catabolism (ie. breakdown of a substance) are typically inducible The substance to be broken down (or a related compound) acts as the inducer Operons involved in anabolism (ie. biosynthesis of a substance) are typically repressible The inhibitor or corepressor is the small molecule that is the product of the operon Inducible vs Repressible Regulation 14-54