Chapter 16: Gene Regulation in Bacteria

Transcription

1 Chapter 16: Gene Regulation in Bacteria Student Learning Objectives Upon completion of this chapter you should be able to: 1. Distinguish between the various mechanisms of transcriptional regulation. 2. Understand the regulation of the lac operon, including the roles of the lac repressor and activator proteins. 3. Understand the regulation of the trp operon, including the molecular details of attenuation. 4. Recognize how repressors and antisense RNAs are used in translational regulation. 5. Recognize how feedback inhibition is used in posttranslational regulation. 6. Know how riboswitches can regulate gene expression of Transcriptional Regulation Gene regulation can occur at a number of levels (Figure 16.1), but the most common is at the transcriptional level. The first section of this chapter examines the variety of means by which genes may be transcriptionally regulated in bacteria. In most cases, transcriptional regulation involves the actions of regulatory proteins that can bind to the DNA and affect the rate of transcription of one or more nearby genes. These proteins include activators and repressors which exert positive and negative control, by small effector proteins which include inducers, inhibitors, and corepressors. It is important that you understand the primary mechanisms of regulation and the terminology associated with each. An overview of these is provided in Figure Outline of Key Terms Gene regulation Constitutive genes Inducible genes Repressible genes Transcriptional regulation Negative control Repressors Positive control Activators Effector molecules Inducer Corepressor Inhibitor Focal Points Common points where regulation of gene expression in bacteria occurs (Figure 16.1) Binding sites on a genetic regulatory protein (Figure 16.2) 183

2 Exercises and Problems For questions 1 to 7, match each regulatory term with its correct definition. 1. Activator 2. Inhibitor 3. Repressor 4. Inducer 5. Negative control 6. Positive control 7. Corepressor a. Gene regulation by repressor proteins. b. Binds to an activator protein and inhibits it from binding to the DNA. c. Gene regulation by activator proteins. d. A protein that binds to the DNA and inhibits transcription. e. A protein that binds to the repressor and causes it to bind to the DNA. f. An effector molecule that increases transcription. g. A protein that increases transcription Regulation of the Lac Operon This section takes a closer look at a specific example of gene regulation in E. coli. Indeed, the lac operon was the first gene regulation system to have its molecular mechanism worked out. Our initial understanding of gene regulation can be traced back to the 1950s and the creative minds of two French scientists: François Jacob and Jacques Monod. They were interested in the phenomenon of enzyme adaptation, which refers to the observation that a particular enzyme appears within a living cell only after the cell has been exposed to the substrate of that enzyme. To investigate this phenomenon, Jacob and Monod focused their attention on lactose metabolism in E. coli, and the rest is history! The lac operon encodes a polycistronic mrna for proteins that are involved in the uptake and metabolism of lactose (See Figure 16.3). The operon can be transcriptionally regulated in two main ways. The first mechanism is one that is inducible and under negative control. This form of regulation involves the lac repressor protein which binds to the operator site. Once bound, the repressor can prevent RNA polymerase from transcribing the genes and so the operon is off. However, this binding is reversible. When allolactose is bound to the repressor, it causes a conformational change that prevents the repressor from binding to the operator. This event induces transcription (See Figure 16.4). The regulation of the lac operon enables E. coli to respond to changes in the level of lactose in it environment (See Figure 16.5). The second mechanism by which the lac operon can be transcriptionally regulated involves an activator protein called CAP. This protein can bind camp and the complex will bind to the CAP site. This stimulates the ability of RNA polymerase to transcribe the lac operon. Glucose inhibits the enzyme adenylyl cyclase which catalyzes the synthesis of camp. Thus, in the presence of glucose, the lac operon is turned off (Refer to Figure 16.8). 184

3 Outline of Key Terms Operon Polycistronic mrna Promoter Terminator Operator Lac operon Lac repressor Allosteric regulation Allosteric site CAP site Catabolite activator protein Enzyme adaptation Merozygote Trans-acting factor Trans-effect cis-acting element cis-effect Catabolite repression Diauxic growth Cyclic AMP (camp) Focal Points Organization of the lac operon (Figure 16.3) Mechanism of induction of the lac operon (Figure 16.4) The cycle of lac operon induction and repression (Figure 16.5) Evidence that the laci gene encodes a diffusible repressor protein (Figure 16.7) The role of the CAP protein in the regulation of the lac operon (Figure 16.8) Exercises and Problems For questions 1 to 11, complete the sentence with the most appropriate term(s): 1. refers to the phenomenon in which the enzymes that metabolize a molecule are made only when that molecule is present in the environment. 2. The two French scientists that elucidated the molecular mechanism of the lac operon are and. 3. The lacy gene encodes the protein, which allows the uptake of lactose into the cell. 4. The lacz gene encodes the enzyme, which cleaves lactose into and. 5. The laci gene encodes the. 6. The operator site is also called, and it is the site where the binds. 7. The promoter of the lac operon is the site where the binds. 8. The lac operon is, meaning that it encodes more than one structural gene. 9. The actual inducer of the lac operon is. 10. Effector molecules bind to a protein s site, which is distinct from the active site. 11. The CAP protein can bind to the CAP site of the lac operon only if it binds first. 185

4 For questions 12 to 18, indicate whether the lac operon will be ON or OFF under the described conditions. 12. Wild-type operon; lactose present; glucose absent 13. Wild-type operon; lactose present; glucose present 14. Mutation in the lac repressor that prevents it from binding to its normal DNA site; lactose absent; glucose absent 15. Mutation in the lac repressor that prevents it from binding to allolactose 16. Mutation in the CAP protein that prevents it from binding to its normal DNA site 17. Mutation in the CAP protein that prevents it from binding to camp 18. Mutation in the lacp region of the operon 16.3 Regulation of the Trp Operon This section examines a second operon in E. coli called the trp operon, which encodes enzymes involved in the synthesis of the amino acid tryptophan. While the lac operon is an inducible operon, the trp operon is a repressible one. The trp operon can be transcriptionally regulated in two main ways. The first mechanism involves the trp repressor. In the absence of tryptophan, this protein is inactive and cannot bind to the operator site. Therefore, RNA polymerase can bind to the promoter and transcribe the genes of the trp operon. When tryptophan levels are high, tryptophan acts as a corepressor. It binds to the trp repressor and activates it. The complex can now bind to the trp operator site to inhibit transcription (See Figures 16.11A and B). The second mechanism by which the trp operon can be transcriptionally regulated is termed attenuation. This mechanism operates under conditions with high levels of tryptophan (See Figure 16.11C). Attenuation involves the formation of a stem-loop structure which causes early termination of transcription (Refer to Figures and 16.13). By studying the genetic regulation of many operons, geneticists have discovered a general trend concerning inducible versus repressible regulation. Inducible operons typically encode catabolic enzymes. The substance to be broken down or a related compound often acts as the inducer. Repressible operons, on the other hand, typically encode anabolic enzymes. The corepressor or inhibitor is commonly the small molecule that is the product of the enzymes biosynthetic activities. Outline of Key Terms Trp repressor Attenuation Attenuator sequence Focal Points Organization of the trp operon (Figure 16.11) Sequence of the trpl mrna produced during attenuation (Figure 16.12) Mechanism of attenuation of the trp operon (Figure 16.13) 184

5 Exercises and Problems For questions 1 to 7, complete the sentence with the most appropriate term(s): 1. The trp operon contains structural genes that encode enzymes involved in the biosynthesis of tryptophan. 2. The trp operon also contains a gene called, which plays a regulatory role. This gene encodes a 14-amino acid protein called the. 3. The trpr gene, which is not part of the trp operon, encodes the. 4. The operator site is also called, and it is the site where the binds. 5. The promoter of the trp operon is the site where the binds. 6. In the trp operon, the corepressor is. 7. Attenuation involves the formation of a stem-loop structure between regions 3 and 4of the attenuator sequence. This stem-loop structure acts as a terminator of transcription. For questions 8 to 10, indicate whether the trp operon will be ON or OFF under the described conditions. 8. Wild-type operon; low levels of tryptophan 9. Wild-type operon; high levels of tryptophan 10. Mutation in the trp repressor that prevents it from binding to tryptophan 11. Why are attenuation systems not found in eukaryotes? 16.4 Translational and Posttranslational Regulation Gene regulation can also occur at the translational and posttranslational levels, although much less commonly than at the transcriptional level. Translational regulation typically involves interactions with the machinery of initiation. The majority of cases involve translational repressors and antisense RNA (Figure 16.14). In posttranslational regulation, the target of regulation is the functional protein. This can occur via feedback inhibition (Figure 16.15) or covalent modifications. Outline of Key Terms Translational regulation Translational regulatory protein Translational repressor Antisense RNA Posttranslational regulation Feedback inhibition Allosteric enzyme Posttranslational covalent modification 184

6 Focal Points Gene regulation via antisense RNA (Figure 16.14) Feedback inhibition (Figure 16.15) Exercises and Problems For the following questions, match the definition with the appropriate term. 1. Any regulatory mechanism that acts on the functional protein. 2. The final product in a metabolic pathway influences an earlier enzyme in the pathway. 3. An RNA strand that is complementary to the mrna strand. 4. Binds to the mrna and prevents it from interacting with the ribosome. 5. An enzyme with two different binding sites, one catalytic and one regulatory. 6. A regulatory mechanism that interacts with the initiation, elongation, or termination of protein synthesis. a. antisense RNA b. translational repressors c. allosteric enzyme d. feedback inhibition e. translational regulation f. posttranslational regulation 16.5 Riboswitches The last decade saw the discovery of riboswitches, which mediate a novel mechanism of gene regulation. A riboswitch involves an RNA molecule that can exist in two different secondary structures, based on the binding of a small molecule. Riboswitches are widespread in bacteria; however, they are also found in archaea, algae, fungi, and plants. A riboswitch can regulate gene expression at several levels, including transcription, translation, and RNA stability (See Table 16.2). Outline of Key Terms Riboswitches Focal Points Riboswitch regulation of transcription in bacteria (Figure 16.16) Riboswitch regulation of translation in bacteria (Figure 16.17) 186

7 Exercises and Problems Characterize the following four statements as True (T) or False (F). If False, change the statement to make it correct. 1. Riboswitches are proteins involved in gene regulation. 2. Riboswitches are found in both prokaryotes and eukaryotes. 3. Riboswitches exist in two alternate secondary structures. 4. Riboswitches regulate gene expression at the levels of transcription, translation, RNA stability, and RNA splicing. 5. Briefly describe how riboswitches differ from antisense RNAs? Chapter Quiz 1. Cyclic AMP is involved in the regulation of which of the following bacterial operons? a. the lac operon b. the trp operon c. Both A and B d. Neither A nor B 2. The operon utilizes attenuation as a regulatory mechanism. a. the lac operon b. the trp operon c. Both A and B d. Neither A nor B 3. Feedback inhibition is a mechanism to regulate gene expression at the level. a. transcriptional b. posttranscriptional c. translational d. posttranslational 4. The lac operon is turned off in the presence of what? a. lactose b. glucose c. arabinose d. any sugar 5. The presence of tryptophan does what to the trp repressor? a. inactivates it b. activates it c. degrades it d. initiates transcription of e. none of the above 187

8 6. A mutation in the lac repressor protein that prevents it from binding to allolactose will cause which of the following? Assume that glucose is not present. a. expression of the lacz gene to be constitutively high b. expression of the lacz gene to be constitutively low c. expression of the lacz gene to be high in the presence of lactose and low in the absence of lactose d. expression of the lacz gene to be low in the presence of lactose and high in the absence of lactose 7. An enzyme that contains both a catalytic site and a regulatory site is called. a. antisense b. inducible c. allosteric d. cis-acting 8. The lac repressor protein binds to the site. a. CAP b. terminator c. promoter d. silencer e. operator 9. The genes required for the breakdown of cellobiose are likely to be I by cellobiose, and the genes required for the synthesis of isoleucine are expected to be II by isoleucine. a. I = induced ; II = repressed b. I = induced ; II = induced c. I = repressed ; II = induced d. I = repressed ; II = repressed 10. Riboswitches have been found in all of the following organisms EXCEPT a. archaea. b. bacteria. c. fungi. d. plants. e. humans. Answer Key for Study Guide Questions This answer key provides the answers to the exercises and chapter quiz for this chapter. Answers in parentheses ( ) represent possible alternate answers to a problem, while answers marked with an asterisk (*) indicate that the response to the question may vary g 2. b 3. d 4. f 5. a 6. c 7. e 188

9 Enzyme adaptation 2. Jacob and Monod 3. lac permease 4. -galactosidase; glucose and galactose 5. lac repressor 6. laco; lac repressor 7. RNA polymerase 8. polycistronic 9. allolactose 10. allosteric site 11. camp 12. ON 13. OFF 14. ON 15. OFF 16. OFF 17. OFF 18. OFF five 2. trpl; leader peptide 3. trp repressor 4. trpo; lac repressor 5. RNA polymerase 6. tryptophan 7. rho-independent 8. ON 9. OFF 10. ON 11. Attenuation requires that transcription and translation be coupled. In eukaryotes, this is not possible because transcription occurs in the nucleus, while translation takes place in the cytoplasm f 2. d 3. a 4. b 5. c 6. e F, are RNA 2. T 3. T 4. T 5. Riboswitches are encoded within the transcript they regulate, and act in cis to control expression of the gene(s) within that transcript, Antisense RNAs, on the other hand, act in trans to regulate the activity of other RNA transcripts. Quiz 1. a 2. b 3. d 4. b 5. b 6. b 7. c 8. e 9. a 10. e 293