1. Gene Regulation in Prokaryotes

2. Outline of Chapter 16 There are many steps in gene expression and regulation can occur at any one of them There are many steps in gene expression and regulation can occur at any one of them Genetic and molecular studies show that most regulation affects the initiation of RNA transcripts Genetic and molecular studies show that most regulation affects the initiation of RNA transcripts Studies of genes for lactose utilization Studies of genes for lactose utilization Negative regulation – blocks transcription Negative regulation – blocks transcription Positive regulation – increases transcription Positive regulation – increases transcription DNA binding proteins acting on RNA polymerase at promoter are main agents of regulation DNA binding proteins acting on RNA polymerase at promoter are main agents of regulation Attenuation of expression – tryptophan pathway Attenuation of expression – tryptophan pathway Gene expression is fine tuned by premature termination of transcription Gene expression is fine tuned by premature termination of transcription

3. Outline of Chapter 16 Global regulatory mechanisms: E. coli’s response to heat shock is an example of the bacterial ability to coordinate the expression of different sets of genes dispersed around the chromosome. Global regulatory mechanisms: E. coli’s response to heat shock is an example of the bacterial ability to coordinate the expression of different sets of genes dispersed around the chromosome. Microarray analysis is an important new tool for detecting changes in gene expression in response to environmental changes Microarray analysis is an important new tool for detecting changes in gene expression in response to environmental changes Comprehensive example: How Vibrio cholerae regulate their virulence genes Comprehensive example: How Vibrio cholerae regulate their virulence genes

4. RNA polymerase is the key enzyme for transcription RNA polymerase involved in three phases of transcription RNA polymerase involved in three phases of transcription Initiation – sigma subunit + core enzyme (two alpha, one beta, and one beta’ subunit) Initiation – sigma subunit + core enzyme (two alpha, one beta, and one beta’ subunit) Binds to promoter, unwinds DNA, begins polymerization of bases complementary to DNA template Binds to promoter, unwinds DNA, begins polymerization of bases complementary to DNA template Elongation – movement away from promoter sigma subunit released, polymerization Elongation – movement away from promoter sigma subunit released, polymerization Termination –signal reached by RNA polymerase Termination –signal reached by RNA polymerase Rho dependent termination – Rho factor recognizes sequence in mRNA, binds to it, and pulls it away from RNA polymerase Rho dependent termination – Rho factor recognizes sequence in mRNA, binds to it, and pulls it away from RNA polymerase Rho independent termination – stem loop structure formed by sequence of 20 bases with a run of 6 or more U’s signals release of RNA polymerase Rho independent termination – stem loop structure formed by sequence of 20 bases with a run of 6 or more U’s signals release of RNA polymerase

5. Fig. 16.2

6. Translation in prokaryotes starts before transcription ends Translation in prokaryotes starts before transcription ends Initiation sites for translation signal ribosomes to bind near 5’ end of mRNA while downstream transcription is still occurring Initiation sites for translation signal ribosomes to bind near 5’ end of mRNA while downstream transcription is still occurring Polycistronic mRNAs often lead to the translation of several genes at the same time from one mRNA transcript Polycistronic mRNAs often lead to the translation of several genes at the same time from one mRNA transcript The regulation of gene expression can occur at many steps The regulation of gene expression can occur at many steps Binding of RNA polymerase to promoter Binding of RNA polymerase to promoter Shift from initiation to elongation Shift from initiation to elongation Release of mRNA at termination Release of mRNA at termination Posttranscriptional stability of mRNA Posttranscriptional stability of mRNA Efficiency of ribosomes to recognize translation initiation sites Efficiency of ribosomes to recognize translation initiation sites Stability of polypeptide product Stability of polypeptide product

7. The utilization of lactose by E. coli: A model system for gene regulation The presence of lactose induces expression of the genes required for lactose utilization The presence of lactose induces expression of the genes required for lactose utilization Induction – stimulation of protein synthesis Induction – stimulation of protein synthesis Inducer – molecule that stimulates synthesis Inducer – molecule that stimulates synthesis Lactose – inducer of genes for lactose utilization Lactose – inducer of genes for lactose utilization 1950s and 1960s – Golden era of bacterial genetics 1950s and 1960s – Golden era of bacterial genetics Advantages of E. coli and lactose utilization system Advantages of E. coli and lactose utilization system Culture large numbers of bacteria allow isolation of rare mutants Culture large numbers of bacteria allow isolation of rare mutants Lactose genes not essential for survival (can use glucose as carbon source) Lactose genes not essential for survival (can use glucose as carbon source) Induction increases expression 1000 fold making mutant identification easy Induction increases expression 1000 fold making mutant identification easy Color changes using  -galactosidase enzyme (e.g., OPNG, X-gal) make measurement of expression levels efficient Color changes using  -galactosidase enzyme (e.g., OPNG, X-gal) make measurement of expression levels efficient

8. Coordinate repression and induction of three genes revealed by studies of lactose-utilization mutants Jacques Monod and Francois Jacob – Pasteur Institute in Paris Jacques Monod and Francois Jacob – Pasteur Institute in Paris Proposed Operon Theory of gene regulation Proposed Operon Theory of gene regulation Single signal can simultaneously regulate expression of several genes that are clustered together on a chromosome and involve the same process Single signal can simultaneously regulate expression of several genes that are clustered together on a chromosome and involve the same process Because genes are clustered, they are transcribed together as single mRNA Because genes are clustered, they are transcribed together as single mRNA Clusters of genes are called Operons Clusters of genes are called Operons

9. Complementation Analysis of mutants identifies lactose utilization genes Monod et al. isolated many Lac - mutants unable to utilize lactose Monod et al. isolated many Lac - mutants unable to utilize lactose Complementation analysis identified three genes (lacZ, lacY, and lacA) in a tightly linked cluster Complementation analysis identified three genes (lacZ, lacY, and lacA) in a tightly linked cluster Fig. 16.5

10. Experimental evidence for repressor protein Isolated mutant in lacI gene Isolated mutant in lacI gene Constitutive mutant – synthesized  - galactosidase and lac permease even in absence of lactose (inducer) Constitutive mutant – synthesized  - galactosidase and lac permease even in absence of lactose (inducer) lacI must be a repressor – cells must need lacI protein product to prevent expression of lacY and lacZ in absence of inducer lacI must be a repressor – cells must need lacI protein product to prevent expression of lacY and lacZ in absence of inducer

11. PaJaMo experiment lacI+, lacZ+ x lacI - lacZ - F - lacI+, lacZ+ x lacI - lacZ - F - Initial synthesis of  - galactosidase stops Initial synthesis of  - galactosidase stops Addition of inducer resumes synthesis Addition of inducer resumes synthesis Conclusion – initial lack of repressor allows synthesis. As lacI is transferred, synthesis stops Conclusion – initial lack of repressor allows synthesis. As lacI is transferred, synthesis stops Repressor stops transcription by binding to operator site near promoter Repressor stops transcription by binding to operator site near promoter Fig. 16.6

12. Inducer releases repressor to trigger enzyme synthesis Addition of lactose inducer caused  – galactosidase synthesis to continue Addition of lactose inducer caused  – galactosidase synthesis to continue Conclusion: Inducer binds to repressor so repressor can not bind to DNA Conclusion: Inducer binds to repressor so repressor can not bind to DNA Allosteric effect - inducer bound to promotor changes conformation of protein so it can not bind to DNA Allosteric effect - inducer bound to promotor changes conformation of protein so it can not bind to DNA

13. Repressor has binding domains for operator and for the inducer Fig. 16.7

14. Changes in the operator can also affect repressor activity Fig. 16.8

15. Proteins act in trans DNA sites act only in cis Trans acting elements can diffuse through cytoplasm and act at target DNA sites on any DNA molecule in cell Trans acting elements can diffuse through cytoplasm and act at target DNA sites on any DNA molecule in cell Cis acting elements can only influence expression of adjacent genes on same DNA molecule Cis acting elements can only influence expression of adjacent genes on same DNA molecule

16. Three experiments elucidate cis and trans acting elements using F’ plasmid Inducible synthesis Inducible synthesis lacI + gene encodes a diffusible element that acts in trans by binding to any operator it encounters regardless of chromosomal location lacI + gene encodes a diffusible element that acts in trans by binding to any operator it encounters regardless of chromosomal location Insert Figure 16.9a here Fig. 16.9 a

17. Noninducible All operator sites (O+) eventually occupied by superrepressor All operator sites (O+) eventually occupied by superrepressor lacI supperrepressor can not bind inducer lacI supperrepressor can not bind inducer lacI s mutant encodes a diffusible element that binds to operator regardless of chromosomal location (trans acting element) lacI s mutant encodes a diffusible element that binds to operator regardless of chromosomal location (trans acting element) Insert Figure 16.9b here Fig. 16.9 b

18. Constitutive Presence of O + plasmid does not compensate for O c mutation on bacterial chromosome Presence of O + plasmid does not compensate for O c mutation on bacterial chromosome Operator is cis acting element Operator is cis acting element Insert Figure 16.9c here Fig. 16.9 c

19. The Operon Theory The players The players lacz, lacY, lacZ genes that split lactose into glucose and galactose lacz, lacY, lacZ genes that split lactose into glucose and galactose Promotor site to which RNA polymerase binds Promotor site to which RNA polymerase binds cis acting operator site cis acting operator site trans-acting repressor that can bind to operator (encoded by lacI gene) trans-acting repressor that can bind to operator (encoded by lacI gene) Inducer that prevents repressor from binding to operator Inducer that prevents repressor from binding to operator Fig. 16.10 a

20. The Operon Theory Repression Repression In absence of lactose, repressor binds to operator which prevents transcription In absence of lactose, repressor binds to operator which prevents transcription Negative regulatory element Negative regulatory element Fig. 16.10 b

21. The Operon Theory Induction Induction Lactose present Lactose present Allolactose binds to repressor. Allolactose binds to repressor. Repressor changes shape and can not bind to operator Repressor changes shape and can not bind to operator RNA polymerase binds to promotor and initiates transcription of polycistronic mRNA RNA polymerase binds to promotor and initiates transcription of polycistronic mRNA Fig. 16.10 c

22. Positive control increases transcription of lacZ, lacY, and lacA cAMP binds to CRP (cAMP receptor protein) when glucose is low cAMP binds to CRP (cAMP receptor protein) when glucose is low CRP binds to regulatory region CRP binds to regulatory region Enhances activity of RNA polymerase at lac promotor Enhances activity of RNA polymerase at lac promotor Fig. 16.11

23. Some positive regulators increase transcription of genes in only one pathway AraC is a positive regulator for all arabinose genes which break down sugar arabinose AraC is a positive regulator for all arabinose genes which break down sugar arabinose Loss of function mutation results in little or no expression of genes Loss of function mutation results in little or no expression of genes Fig. 16.12

24. Molecular studies help fill in details of control mechanisms Radioactive tag attached to lac repressor Radioactive tag attached to lac repressor Repressor from lacI + cells purified and mixed with operator DNA, cosediment occurred Repressor from lacI + cells purified and mixed with operator DNA, cosediment occurred Repressor from lacI + mixed with mutant operator DNA, no cosediment occurred Repressor from lacI + mixed with mutant operator DNA, no cosediment occurred Fig. 16.13

25. Many DNA-Binding proteins contain a helix-turn-helix motif Two α-helical regions separated by a turn in the protein structure Two α-helical regions separated by a turn in the protein structure Helix-turn-helix motif fits into major groove of DNA Helix-turn-helix motif fits into major groove of DNA Most repressor proteins Most repressor proteins Fig. 16.14 a

26. Specific amino acids in the a-helix determine the binding specificity of repressor proteins Hybrid 434-P22 repressor engineered to have amino acid sequence that will bind to bacterial virus 434 and bacteriophage P22 Hybrid 434-P22 repressor engineered to have amino acid sequence that will bind to bacterial virus 434 and bacteriophage P22 Fig. 16.14 b

27. Most regulatory proteins are oligomeric  More than one binding domain  DNase footprint identifies binding region  DNase cannot digest protein covered sites Fig. 16.15 a

28. The looping of DNA is a common feature of regulatory proteins AraC acts as both a repressor and activator AraC acts as both a repressor and activator No arabinose No arabinose Binding to araO and Binding to araO and araI 1 causes looping and prevents RNA from transcribing araI 1 causes looping and prevents RNA from transcribing Arabinose present Arabinose present AraC binds to araI 1 and araI 2 bot not to araO. RNA polymerase interacts with araC at the araI sites and transcribes genes AraC binds to araI 1 and araI 2 bot not to araO. RNA polymerase interacts with araC at the araI sites and transcribes genes Fig. 16.16

29. How regulatory proteins interact with RNA polymerase Negative regulators (lac repressor) Negative regulators (lac repressor) Physically block DNA-binding sites of RNA polymerase Physically block DNA-binding sites of RNA polymerase Positive regulators Positive regulators Establish physical contact with RNA polymerase enhancing enzyme’s ability to initiate transcription Establish physical contact with RNA polymerase enhancing enzyme’s ability to initiate transcription

30. Using the lacZ gene as a reporter of gene expression Reporter gene – protein encoding gene whose expression in the cell is quantifiable by techniques of protein detection. Reporter gene – protein encoding gene whose expression in the cell is quantifiable by techniques of protein detection. Fusion of reporter gene to cis acting regulatory regions allows assessment gene activity by monitoring amount of reporter gene product Fusion of reporter gene to cis acting regulatory regions allows assessment gene activity by monitoring amount of reporter gene product

31. Fusion used to perform genetic studies of the regulatory region of gene X Fig. 16.18 a

32. Creating a collection of lacZ insertions in the chromosome Fig. 16.18 b

33. Use of a fusion to overproduce a gene product Fig. 16.18 c

34. The attenuation of gene expression: Fine tuning of the trp operon through termination of transcription The presence of tryptophan activates a repressor of the trp operon The presence of tryptophan activates a repressor of the trp operon trpR gene produces repressor trpR gene produces repressor Corepressor – tryptophan binds to trp repressor allowing it to bind to operator DNA and inhibit transcription Corepressor – tryptophan binds to trp repressor allowing it to bind to operator DNA and inhibit transcription

36. Termination of transcription fine tunes regulation of trp operon trpR - mutants are not constitutive trpR - mutants are not constitutive Repressor independent change in trp expression Repressor independent change in trp expression Two alternative transcripts lead to different transcriptional outcomes Two alternative transcripts lead to different transcriptional outcomes Leader sequence can fold in two different stable conformations Leader sequence can fold in two different stable conformations Tryptophan present – ribosome moves quickly past codons in leader allowing stem-loop to form terminating transcription Tryptophan present – ribosome moves quickly past codons in leader allowing stem-loop to form terminating transcription Tryptophan absent – ribosome stalls allowing normal stem loop structure to form and transcription proceeds normally Tryptophan absent – ribosome stalls allowing normal stem loop structure to form and transcription proceeds normally

38. Global regulatory mechanisms coordinate the expression of many genes Normal sigma factor (  70 ) binds to RNA polymerase and recognizes sequence in promoter to initiate transcription Normal sigma factor (  70 ) binds to RNA polymerase and recognizes sequence in promoter to initiate transcription Heat shock disables  70 Heat shock disables  70 Product of rpoH gene,  32 binds to sequence in promoter of heat shock genes when heat stressed and starts transcription Product of rpoH gene,  32 binds to sequence in promoter of heat shock genes when heat stressed and starts transcription Fig. 16.21 a

39. Factors influencing increase in  32 activity after heat shock Increase in transcription of the rpoH gene Increase in transcription of the rpoH gene Increase in the translation of  32 mRNA stemming from greater stability of rpoH mRNA Increase in the translation of  32 mRNA stemming from greater stability of rpoH mRNA Increase in the stability and activity of the  32 protein. Chaperones DnaJ/K bind and inhibit  32 under normal conditions. At high temperature, binding to  32 does not occur and more  32 is free to associate with RNA polymerase. Increase in the stability and activity of the  32 protein. Chaperones DnaJ/K bind and inhibit  32 under normal conditions. At high temperature, binding to  32 does not occur and more  32 is free to associate with RNA polymerase. Inactivity of  70 decreases competition with  32 to form RNA polymerase holoenzyme Inactivity of  70 decreases competition with  32 to form RNA polymerase holoenzyme

40. What enables transcription of  32 during heat shock? Normal temperatures, rpoH gene (encodes  32 ) has promoter sequence recognized by  70 which starts transcription Normal temperatures, rpoH gene (encodes  32 ) has promoter sequence recognized by  70 which starts transcription High temperatures (no  70 ) a different promoter sequence of the rpoH gene is recognized by a different sigma factor,  24 High temperatures (no  70 ) a different promoter sequence of the rpoH gene is recognized by a different sigma factor,  24

41. Summary E. coli’s heat shock response is controlled by alternative sigma factors that recognize different promoter sequences E. coli’s heat shock response is controlled by alternative sigma factors that recognize different promoter sequences Alternative sigma factors bind to RNA polymerase as temperatures change to start transcription of heat shock proteins Alternative sigma factors bind to RNA polymerase as temperatures change to start transcription of heat shock proteins The induction of alternative sigma factors that recognize different promoter sequences serve as global control regulatory mechanisms in E. coli and many other bacteria The induction of alternative sigma factors that recognize different promoter sequences serve as global control regulatory mechanisms in E. coli and many other bacteria

42. Microarrays – a tool for uncovering changes in gene expression Cellular responses to global environmental changes can be measured by microarray analysis of mRNA isolated from cultures grown in different environmental conditions Cellular responses to global environmental changes can be measured by microarray analysis of mRNA isolated from cultures grown in different environmental conditions Comparisons of wild-type cultures with strains containing mutations in key regulatory regions help identify genes and regulatory elements involved in response to specific environmental changes Comparisons of wild-type cultures with strains containing mutations in key regulatory regions help identify genes and regulatory elements involved in response to specific environmental changes

43. Fig. 1.13

44. Regulation of Virulence Genes in V. cholerae Bacterial agents of cholera sense changes in environment and transmit signals to regulators that initiate, enhance, diminish, or repress expression of various genes. Bacterial agents of cholera sense changes in environment and transmit signals to regulators that initiate, enhance, diminish, or repress expression of various genes. Three regulatory proteins – ToxR, ToxS, and ToxT – turn on the genes for virulence Three regulatory proteins – ToxR, ToxS, and ToxT – turn on the genes for virulence

45. Experiments generate model for regulation of virulence genes in V. cholerae Cloned two genes encoding subunits of cholera toxin: ctxA and ctxB Cloned two genes encoding subunits of cholera toxin: ctxA and ctxB Made ctxA-lacZ reporter gene fusion Made ctxA-lacZ reporter gene fusion Created vector library of V. cholerae genomic DNA Created vector library of V. cholerae genomic DNA Used E. coli to perform genetic manipulations Used E. coli to perform genetic manipulations

46. Isolated a gene that regulates expression of ctx operon Isolated a gene that regulates expression of ctx operon Transformed E. coli containing ctx-lacZ construct with clones containing V. cholerae DNA Transformed E. coli containing ctx-lacZ construct with clones containing V. cholerae DNA Clones that contain a positive regulator should turn on ctx-lacZ construct Clones that contain a positive regulator should turn on ctx-lacZ construct Identified ToxR, a transmembrane protein Identified ToxR, a transmembrane protein Identified ToxS, helps ToxR form dimers which helps it bind to DNA Identified ToxS, helps ToxR form dimers which helps it bind to DNA What genes does ToxR regulate? What genes does ToxR regulate? Gene fusions created to constitutive promoter Gene fusions created to constitutive promoter Fusion introduced into strains of V. cholerae with lacZ randomly inserted around the genome Fusion introduced into strains of V. cholerae with lacZ randomly inserted around the genome Identified intermediate regulator gene ToxT, a transcriptional activator that binds to promoters of many genes, including ctx Identified intermediate regulator gene ToxT, a transcriptional activator that binds to promoters of many genes, including ctx ToxR/S or ToxT can activate the ctx genes that produce toxin ToxR/S or ToxT can activate the ctx genes that produce toxin ToxT alone activates additional virulence genes which encode pili and other proteins ToxT alone activates additional virulence genes which encode pili and other proteins Transcription of ToxT is regulated by ToxR/S Transcription of ToxT is regulated by ToxR/S

47. Fig. 16.22

48. Unanswered Questions Remain Why is there a cascade (ToxR and ToxT) of regulatory factors? Why is there a cascade (ToxR and ToxT) of regulatory factors? What DNA sequence in the promoters does ToxR recognize? What DNA sequence in the promoters does ToxR recognize? What is the signal that’s makes the cholera bacteria start to colonize the small intestine? What is the signal that’s makes the cholera bacteria start to colonize the small intestine? How does ToxR regulatory protein find binding sites on the chromosome? How does ToxR regulatory protein find binding sites on the chromosome?