1. Copyright © 2009 Pearson Education Inc., publishing as Pearson Benjamin Cummings Brock Biology of Microorganisms Twelfth Edition Madigan / Martinko Dunlap / Clark Regulation of Gene Expression Chapter 9

2. Copyright © 2009 Pearson Education Inc., publishing as Pearson Benjamin Cummings I. Overview of Regulation  9.1Major Modes of Regulation

3. Copyright © 2009 Pearson Education Inc., publishing as Pearson Benjamin Cummings 9.1 Major Modes of Regulation  Gene expression: transcription of gene into mRNA followed by translation of mRNA into a protein  Most proteins are enzymes that carry out biochemical reactions essential for cell growth  Constitutive proteins are needed at the same level all the time  Microbial genomes encode many more proteins than are present at any one time  Regulation is important in all cells and helps conserve energy and resources

4. Copyright © 2009 Pearson Education Inc., publishing as Pearson Benjamin Cummings 9.1 Major Modes of Regulation  Two major levels of regulation in the cell  One controls the activity of preexisting enzymes  Posttranslational regulation  Very rapid process (seconds)  One controls the amount of an enzyme  Regulate level of transcription  Regulate translation  Slower process (minutes)

5. Copyright © 2009 Pearson Education Inc., publishing as Pearson Benjamin Cummings An Overview of Mechanisms of Regulation Figure 9.1

6. Copyright © 2009 Pearson Education Inc., publishing as Pearson Benjamin Cummings II. DNA-Binding Proteins and Regulation of Transcription  9.2DNA-Binding Proteins  9.3Negative Control of Transcription: Repression and Induction  9.4Positive Control of Transcription

7. Copyright © 2009 Pearson Education Inc., publishing as Pearson Benjamin Cummings 9.2 DNA-Binding Proteins  R ecall that mRNA transcripts generally have a short half-life  Prevents the production of unneeded proteins  Regulation of transcription typically requires proteins that can bind to DNA  Small molecules influence the binding of regulatory proteins to DNA  Proteins actually regulate transcription

8. Copyright © 2009 Pearson Education Inc., publishing as Pearson Benjamin Cummings 9.2 DNA-Binding Proteins  Most DNA-binding proteins interact with DNA in a sequence-specific manner  Specificity provided by interactions between amino acid side chains and chemical groups on the bases and sugar-phosphate backbone of DNA  Major groove of DNA is the main site of protein binding  Inverted repeats frequently are binding site for regulatory proteins

9. Copyright © 2009 Pearson Education Inc., publishing as Pearson Benjamin Cummings 9.2 DNA-Binding Proteins  Homodimeric proteins: proteins composed of two identical polypeptides  Protein dimers interact with inverted repeats on DNA  Each of the polypeptides binds to one inverted repeat

10. Copyright © 2009 Pearson Education Inc., publishing as Pearson Benjamin Cummings DNA-Binding Proteins Figure 9.2

11. Copyright © 2009 Pearson Education Inc., publishing as Pearson Benjamin Cummings 9.2 DNA-Binding Proteins  Several classes of protein domains are critical for proper binding of proteins to DNA  Helix-turn-helix  First helix is the recognition helix  Second helix is the stabilizing helix  Many different DNA-binding proteins from Bacteria contain helix-turn-helix  lac and trp repressors of E. coli

12. Copyright © 2009 Pearson Education Inc., publishing as Pearson Benjamin Cummings The Helix-Turn-Helix Structure of Some DNA-Binding Proteins Figure 9.3

13. Copyright © 2009 Pearson Education Inc., publishing as Pearson Benjamin Cummings 9.2 DNA-Binding Proteins  Classes of Protein Domains  Zinc finger  Protein structure that binds a zinc ion  Typically two or three zinc fingers on proteins that use them for DNA binding  Leucine zipper  Leucine residues are spaced every seven amino acids  Does not interact directly with DNA

14. Copyright © 2009 Pearson Education Inc., publishing as Pearson Benjamin Cummings Models of Protein Substructures in DNA-Binding Proteins Figure 9.4

15. Copyright © 2009 Pearson Education Inc., publishing as Pearson Benjamin Cummings 9.2 DNA-Binding Proteins  Multiple outcomes after DNA binding are possible 1)DNA-binding protein may catalyze a specific reaction on the DNA molecule (i.e., transcription by RNA polymerase) 2)The binding event can block transcription (negative regulation) 3)The binding event can activate transcription (positive regulation)

16. Copyright © 2009 Pearson Education Inc., publishing as Pearson Benjamin Cummings 9.3 Negative Control of Transcription: Repression and Induction  Several mechanisms for controlling gene expression in bacteria  These systems are greatly influenced by environment in which the organism is growing  Presence or absence of specific small molecules  Interaction between small molecules and DNA-binding proteins result in control of transcription or translation

17. Copyright © 2009 Pearson Education Inc., publishing as Pearson Benjamin Cummings  Negative control: a regulatory mechanism that stops transcription  Repression: preventing the synthesis of an enzyme in response to a signal  Enzymes affected by any repression make up a small fraction of total proteins in the cell  Typically affects anabolic enzymes (i.e., arginine biosynthesis) 9.3 Negative Control of Transcription: Repression and Induction

18. Copyright © 2009 Pearson Education Inc., publishing as Pearson Benjamin Cummings Enzyme Repression Figure 9.5

19. Copyright © 2009 Pearson Education Inc., publishing as Pearson Benjamin Cummings Operons Repression Animation: Operons: Repression Animation: Operons: Repression

20. Copyright © 2009 Pearson Education Inc., publishing as Pearson Benjamin Cummings 9.3 Negative Control of Transcription: Repression and Induction  Negative Control (cont’d)  Induction: production of an enzyme in response to a signal  Typically affects catabolic enzymes (i.e., lac operon)  Enzymes are synthesized only when they are needed  no wasted energy

21. Copyright © 2009 Pearson Education Inc., publishing as Pearson Benjamin Cummings Enzyme Induction Figure 9.6

22. Copyright © 2009 Pearson Education Inc., publishing as Pearson Benjamin Cummings Operons Induction Animation: Operons: Induction Animation: Operons: Induction

23. Copyright © 2009 Pearson Education Inc., publishing as Pearson Benjamin Cummings 9.3 Negative Control of Transcription: Repression and Induction  Inducer: substance that induces enzyme synthesis  Corepressor: substance that represses enzyme synthesis  Effectors: collective term for inducers and repressors  Effectors affect transcription indirectly by binding to specific DNA-binding proteins  Repressor molecules bind to an allosteric repressor protein  Allosteric repressor becomes active and binds to region of DNA near promoter called the operator

24. Copyright © 2009 Pearson Education Inc., publishing as Pearson Benjamin Cummings 9.3 Negative Control of Transcription: Repression and Induction  Operon: cluster of genes arranged in a linear fashion whose expression is under control of a single operator  Operator is located downstream of the promoter  Transcription is physically blocked when repressor binds to operator  Enzyme induction can also be controlled by a repressor  Addition of inducer inactivates repressor and transcription can proceed  Repressor’s role is inhibitory so it is called negative control

25. Copyright © 2009 Pearson Education Inc., publishing as Pearson Benjamin Cummings The Process of Enzyme Repression Figure 9.7

26. Copyright © 2009 Pearson Education Inc., publishing as Pearson Benjamin Cummings The Process of Enzyme Induction Figure 9.8

27. Copyright © 2009 Pearson Education Inc., publishing as Pearson Benjamin Cummings 9.4 Positive Control of Transcription  Positive control: regulator protein activates the binding of RNA polymerase to DNA  Maltose catabolism in E. coli  Maltose activator protein cannot bind to DNA unless it first binds maltose  Activator proteins bind specifically to certain DNA sequence  Called activator binding site, not operator

28. Copyright © 2009 Pearson Education Inc., publishing as Pearson Benjamin Cummings 9.4 Positive Control of Transcription  Promoters of positively controlled operons only weakly bind RNA polymerase  Activator protein helps RNA polymerase recognize promoter  May cause a change in DNA structure  May interact directly with RNA polymerase  Activator-binding site may be close to the promoter or several hundred base pairs away

29. Copyright © 2009 Pearson Education Inc., publishing as Pearson Benjamin Cummings Computer Model of Positive Regulatory Protein and DNA Figure 9.10

30. Copyright © 2009 Pearson Education Inc., publishing as Pearson Benjamin Cummings Activator Protein Interactions with RNA Polymerase Figure 9.11

31. Copyright © 2009 Pearson Education Inc., publishing as Pearson Benjamin Cummings 9.4 Positive Control of Transcription  Genes for maltose are spread out over the chromosome in several operons  Each operon has an activator-binding site  Multiple operons controlled by the same regulatory protein are called a regulon  Regulons also exist for negatively controlled systems

32. Copyright © 2009 Pearson Education Inc., publishing as Pearson Benjamin Cummings III. Sensing and Signal Transduction  9.5Two-Component Regulatory Systems  9.6Quorum Sensing  9.7Regulation of Chemotaxis  9.8Control of Transcription in Archaea

33. Copyright © 2009 Pearson Education Inc., publishing as Pearson Benjamin Cummings 9.5 Two-Component Regulatory Systems  Prokaryotes regulate cellular metabolism in response to environmental fluctuations  External signal is not always transmitted directly to the target to be regulated  Signal transduction: External signal can be detected by a sensor and transmitted to regulatory machinery  Most signal transduction systems are two-component regulatory systems

34. Copyright © 2009 Pearson Education Inc., publishing as Pearson Benjamin Cummings 9.5 Two-Component Regulatory Systems  Two-component regulatory systems  Made up of two different proteins  Sensor kinase: (cytoplasmic membrane) detects environmental signal and autophosphorylates  Response regulator: (cytoplasm) DNA-binding protein that regulates transcription  Absent in bacteria that live as parasites of higher organisms

35. Copyright © 2009 Pearson Education Inc., publishing as Pearson Benjamin Cummings 9.5 Two-Component Regulatory Systems  Almost 50 different two-component systems in E. coli  Examples include phosphate assimilation, nitrogen metabolism, and osmotic pressure response  Some signal transduction systems have multiple regulatory elements  Some Archaea also have two-component regulatory systems

36. Copyright © 2009 Pearson Education Inc., publishing as Pearson Benjamin Cummings Control of Gene Expression by a Two-Component System Figure 9.12

37. Copyright © 2009 Pearson Education Inc., publishing as Pearson Benjamin Cummings Examples of Two-Component Regulatory Systems

38. Copyright © 2009 Pearson Education Inc., publishing as Pearson Benjamin Cummings 9.6 Quorum Sensing  Prokaryotes can respond to the presence of other cells of the same species  Quorum sensing: mechanism by which bacteria assess their population density  Ensures sufficient number of cells are present before initiating a response that requires a certain cell density to have an effect (i.e., toxin production in pathogenic bacterium)

39. Copyright © 2009 Pearson Education Inc., publishing as Pearson Benjamin Cummings 9.6 Quorum Sensing  Each species of bacterium produces a specific autoinducer molecule  Diffuses freely across the cell envelope  Reaches high concentrations inside cell only if many cells are near  Binds to specific activator protein and triggers transcription of specific genes

40. Copyright © 2009 Pearson Education Inc., publishing as Pearson Benjamin Cummings 9.6 Quorum Sensing  Several different classes of autoinducers  Acyl homoserine lactone was the first autoinducer to be identified  Quorum sensing first discovered as mechanism regulating light production in bacteria including V. fischeri  Lux operon encodes bioluminescence

41. Copyright © 2009 Pearson Education Inc., publishing as Pearson Benjamin Cummings Quorum Sensing Figure 9.13

42. Copyright © 2009 Pearson Education Inc., publishing as Pearson Benjamin Cummings Bioluminescent Bacteria Producing the Enzyme Luciferase Figure 9.14

43. Copyright © 2009 Pearson Education Inc., publishing as Pearson Benjamin Cummings 9.6 Quorum Sensing  Examples of Quorum Sensing  P. aeruginosa switches from free living to growing as a biofilm  Virulence factors of S. aureus  Quorum sensing is present in some microbial eukaryotes  Quorum sensing likely exists in Archaea

44. Copyright © 2009 Pearson Education Inc., publishing as Pearson Benjamin Cummings 9.7 Regulation of Chemotaxis  Modified two-component system used in chemotaxis to  Sense temporal changes in attractants or repellents  Regulate flagellar rotation  Three main steps 1)Response to signal 2)Controlling flagellar rotation 3)Adaptation

45. Copyright © 2009 Pearson Education Inc., publishing as Pearson Benjamin Cummings 9.7 Regulation of Chemotaxis  Step1: Response to Signal  Sensory proteins in cytoplasmic membrane sense presence of attractants and repellents  Methyl-accepting chemotaxis proteins (MCPs)  MCPs bind attractant or repellent and initiate interactions that eventually affect flagellar rotation

46. Copyright © 2009 Pearson Education Inc., publishing as Pearson Benjamin Cummings 9.7 Regulation of Chemotaxis  Step 2: Controlling Flagellar Rotation  Controlled by CheY protein  CheY results in counterclockwise rotation and runs  CheY-P results in clockwise rotation and tumbling

47. Copyright © 2009 Pearson Education Inc., publishing as Pearson Benjamin Cummings 9.7 Regulation of Chemotaxis  Step 3: Adaptation  Feedback loop  Allows the system to reset itself to continue to sense the presence of a signal  Involves modification of MCPs

48. Copyright © 2009 Pearson Education Inc., publishing as Pearson Benjamin Cummings Interactions of MCPs, Che Proteins and the Flagellar Motor Figure 9.15

49. Copyright © 2009 Pearson Education Inc., publishing as Pearson Benjamin Cummings 9.8 Control of Transcription in Archaea  Archaea use DNA-binding proteins to control transcription  More closely resembles control by Bacteria than Eukarya

50. Copyright © 2009 Pearson Education Inc., publishing as Pearson Benjamin Cummings Repression of Genes for Nitrogen Metabolism in Archaea Figure 9.16

51. Copyright © 2009 Pearson Education Inc., publishing as Pearson Benjamin Cummings IV. Global Regulatory Mechanisms  9.9Global Control and the lac Operon  9.10The Stringent Response  9.11Other Global Control Networks

52. Copyright © 2009 Pearson Education Inc., publishing as Pearson Benjamin Cummings 9.9 Global Control and the lac Operon  Global control systems: regulate expression of many different genes simultaneously  Catabolite repression is an example of global control  Synthesis of unrelated catabolic enzymes is repressed if glucose is present in growth medium  lac operon is under control of catabolite repression  Ensures the “best” carbon and energy source is used first  Diauxic growth: two exponential growth phases

53. Copyright © 2009 Pearson Education Inc., publishing as Pearson Benjamin Cummings Diauxic Growth of E. coli on Glucose and Lactose Figure 9.17

54. Copyright © 2009 Pearson Education Inc., publishing as Pearson Benjamin Cummings 9.9 Global Control and the lac Operon  Dozens of catabolic operons affected by catabolite repression  Enzymes for degrading lactose, maltose, and other common carbon sources  Flagellar genes are also controlled by catabolite repression  No need to swim in search of nutrients

55. Copyright © 2009 Pearson Education Inc., publishing as Pearson Benjamin Cummings 9.10 The Stringent Response  In natural environments nutrients appear/disappear rapidly  Stringent response: global control mechanism triggered by amino acid starvation  Triggered by (p)ppGpp  Alarmones: produced by RelA to signal amino acid starvation  Stringent response only in Bacteria  Achieves balance within the call between protein production and protein requirements

56. Copyright © 2009 Pearson Education Inc., publishing as Pearson Benjamin Cummings The Stringent Response Figure 9.20ab

57. Copyright © 2009 Pearson Education Inc., publishing as Pearson Benjamin Cummings The Stringent Response Figure 9.20cd

58. Copyright © 2009 Pearson Education Inc., publishing as Pearson Benjamin Cummings 9.11 Other Global Control Networks  Several other global control systems in E. coli  Heat shock response: largely controlled by alternative sigma factors  Heat shock proteins: counteract damage of denatured proteins and help cell recover from temperature stress  Very ancient proteins  Heat shock response also occurs in Archaea

59. Copyright © 2009 Pearson Education Inc., publishing as Pearson Benjamin Cummings Control of Heat Shock in Escherichia coli Figure 9.21

60. Copyright © 2009 Pearson Education Inc., publishing as Pearson Benjamin Cummings Examples of Global Control Systems Known in E. coli

61. Copyright © 2009 Pearson Education Inc., publishing as Pearson Benjamin Cummings V. Regulation of Development in Model Bacteria  9.12Sporulation in Bacillus  9.13Caulobacter Differentiation

62. Copyright © 2009 Pearson Education Inc., publishing as Pearson Benjamin Cummings 9.12 Sporulation in Bacillus  Regulation of development in model bacteria  Some prokaryotes display the basic principle of differentiation  Endospore formation in Bacillus  Form inside mother cell  Triggered by adverse external conditions (i.e., starvation or dessication)

63. Copyright © 2009 Pearson Education Inc., publishing as Pearson Benjamin Cummings Control of Endospore Formation in Bacillus Figure 9.22

64. Copyright © 2009 Pearson Education Inc., publishing as Pearson Benjamin Cummings 9.13 Caulobacter Differentiation  Caulobacter provide another example of differentiation  Two forms of cells  Swarmer cells: dispersal role  Stalked cells: reproductive role  Many details are still uncertain  External stimuli and internal factors do play a role in affecting life cycle

65. Copyright © 2009 Pearson Education Inc., publishing as Pearson Benjamin Cummings Cell Cycle Regulation in Caulobacter Figure 9.23

66. Copyright © 2009 Pearson Education Inc., publishing as Pearson Benjamin Cummings VI. RNA-Based Regulation  9.14RNA Regulation and Antisense RNA  9.15Riboswitches  9.16Attenuation

67. Copyright © 2009 Pearson Education Inc., publishing as Pearson Benjamin Cummings 9.14 RNA Regulation and Antisense RNA  Regulatory RNA molecules exert their effects by base pairing with mRNA  Double-stranded region prevents translation of mRNA  These small RNAs (~100 nucleotides) are called antisense RNAs  Each antisense RNA can regulate multiple mRNAs  Transcription of antisense RNA is enhanced when its target genes need to be turned off  Some antisense RNA actually enhance translation

68. Copyright © 2009 Pearson Education Inc., publishing as Pearson Benjamin Cummings Regulation by Antisense RNA Figure 9.24

69. Copyright © 2009 Pearson Education Inc., publishing as Pearson Benjamin Cummings 9.15 Riboswitches  Riboswitches: RNA domains in an mRNA molecule that can bind small molecules to control translation of mRNA  Located at 5′ end of mRNA  Binding results from folding of RNA into a 3-D structure  Similar to a protein recognizing a substrate  Riboswitch control is analogous to negative control  Found in some bacteria, fungi, and plants

70. Copyright © 2009 Pearson Education Inc., publishing as Pearson Benjamin Cummings Regulation by Riboswitch Figure 9.25

71. Copyright © 2009 Pearson Education Inc., publishing as Pearson Benjamin Cummings 9.16 Attenuation  Transcriptional control that functions by premature termination of mRNA synthesis  Control exerted after the initiation of transcription, but before its completion  First example was the tryptophan operon in E. coli  mRNA stem-loop structure and synthesis of leader peptide are determining factors in attenuation  Genomic evidence suggests attenuation exists in Archaea

72. Copyright © 2009 Pearson Education Inc., publishing as Pearson Benjamin Cummings Attenuation and the Leader Peptide Figure 9.26

73. Copyright © 2009 Pearson Education Inc., publishing as Pearson Benjamin Cummings Mechanism of Attenuation Figure 9.27

74. Copyright © 2009 Pearson Education Inc., publishing as Pearson Benjamin Cummings Mechanism of Attenuation Figure 9.27