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2 Pearson Education Limited Edinburgh Gate Harlow Essex CM20 2JE England and Associated Companies throughout the world Visit us on the World Wide Web at: Pearson Education Limited 2014 All rights reserved. No part of this publication may be reproduced, stored in a retrieval system, or transmitted in any form or by any means, electronic, mechanical, photocopying, recording or otherwise, without either the prior written permission of the publisher or a licence permitting restricted copying in the United Kingdom issued by the Copyright Licensing Agency Ltd, Saffron House, 6 10 Kirby Street, London EC1N 8TS. All trademarks used herein are the property of their respective owners. The use of any trademark in this text does not vest in the author or publisher any trademark ownership rights in such trademarks, nor does the use of such trademarks imply any affiliation with or endorsement of this book by such owners. ISBN 10: ISBN 13: British Library Cataloguing-in-Publication Data A catalogue record for this book is available from the British Library Printed in the United States of America

3 is needed. As it turns out, this is a common type of control. In most cases, the activity of key regulatory proteins is controlled by post-translational modifications. check your understanding C Y U If you understand that... Negative control occurs when something must be taken away for transcription to occur. The lac operon repressor exerts negative control over three protein-coding genes by binding to the operator site in DNA, near the promoter. For transcription to occur in the lac operon, an inducer molecule (a derivative of lactose) must bind to the repressor, causing it to change shape and release from the operator. The lac operon is not transcribed when glucose is available because glucose prevents lactose transport into the cell. You should be able to Explain why lactose should induce transcription of the lac operon. 2. Diagram the lac operon, showing the relative positions of the operator, the promoter, and the three proteincoding genes; indicate what is happening at the operon in the absence of lactose and in the presence of lactose. 4 working on a project for a laboratory course were the first to uncover it. This operon contains three genes that allow E. coli to use the sugar arabinose. Arabinose is found in many plant cell walls. When you eat vegetables, arabinose is available to the bacteria that inhabit your gut. Without arabinose in the environment, the ara operon is not transcribed. But when arabinose is present, transcription of the ara operon is turned on by an activator protein called AraC. The ara operon and an adjacent gene, arac, that codes for the arac activator are shown in Figure 9. Figure 10 outlines how AraC controls the ara operon. The AraC protein is allosterically regulated by arabinose. When bound to arabinose, two copies of the AraC protein attach to a regulatory sequence of DNA called the ara initiator that lies just upstream of the promoter (see Figure 10a). Once AraC is bound to DNA, it can also bind to RNA polymerase. This interaction between AraC and the RNA polymerase helps to dock the polymerase to the promoter and accelerate the initiation of transcription. Continued work on the ara operon revealed a surprise AraC is both an activator and a repressor. In the absence of arabinose, the two copies of the AraC protein remain together; but while one arac copy remains bound to the initiator, the other copy now binds to a different regulatory site in DNA, the ara operator, as shown in Figure 10b. In this configuration, AraC works as a repressor to prevent the transcription of both the ara operon and the arac gene. If you understand positive control by the AraC protein, you should be able to predict the effect of a mutation that removes the part of AraC that binds to RNA polymerase. 5 Positive Control of Transcription Positive control is an important way of controlling transcription. In positive control, an activator protein binds to a regulatory sequence in DNA when genes are turned on. When bound to DNA, the activator interacts with RNA polymerase to increase the rate of initiating transcription (see Figure 6b). The ara operon provides an important example of positive control and of the process of science. The ara operon wasn t discovered in the laboratory of a famous scientist. Instead, students global gene regulation A theme of this chapter is that cells respond to changing environments. To compete for resources, bacteria must be able to coordinate the expression of large sets of genes. As you ve seen for the lac and ara operons, an effective way to express sets of genes together is to group them into an operon and transcribe them into a single mrna. But there are limits to the size of operons. How can bacterial cells manage responses that require the expression of dozens or even hundreds of genes? ara operon Regulatory sequences arac Operator arac promoter (PC) Initiator arabad promoter (PBAD) arab araa Enzymes required for arabinose metabolism AraC (regulatory protein) Figure 9 The ara Operon, Regulatory Sequences, and arac Gene. 442 arad Pg. No. 10

4 (a) AraC protein is an activator when bound to arabinose. Promoter TRANSCRIPTION Initiator arab AraC dimer Arabinose araa arad RNA polymerase (b) AraC protein is a repressor when arabinose is absent. Initiator PBAD arab araa arad AraC dimer PC Operator arac Figure 10 Positive and Negative Regulation of the ara Operon. Global gene regulation is the coordinated regulation of many genes. You already learned that alternative sigma proteins provide one way for bacteria to turn on large numbers of genes in response to environmental change. But there are other means of global gene regulation, such as grouping genes into a regulon a set of separate genes or operons that contain the same regulatory sequences and that are controlled by a single type of regulatory protein. Regulons allow bacteria to respond to challenges that include shortages of nutrients, sudden changes in temperature, exposure to radiation, or shifts in habitat. Let s explore how regulons work in general, and then look at two specific examples. A general strategy for controlling regulon genes is shown in Figure 11. In this example, the regulon consists of many genes that are scattered across the genome. All of these genes are controlled by the same type of repressor protein that binds to the same operator sequences near the promoter of each gene. When an environmental change triggers the removal of the repressor protein from all the operators, every gene in the regulon is transcribed. Regulons can be under negative control by a repressor protein or positive control by an activator protein. The regulon in Figure 11 is under negative control. The SOS response regulon works exactly this way to allow bacterial cells to repair extensive damage to DNA that can occur when cells are exposed to ultraviolet light, other types of radiation, or some chemicals. Damaged DNA sets off an SOS signal that induces the transcription of more than 40 genes that code for DNA repair enzymes and for DNA polymerases that can use damaged DNA as a template. Without the SOS response, bacteria with massive DNA damage would face almost certain death. The ToxR regulon of Vibrio cholera the bacterium that causes cholera is under positive control. This regulon allows V. cholera to colonize the human gut and to produce toxins that cause diarrhea. Cholera kills 120,000 people each year and sickens as many as 18 million. ToxR regulon genes are inactive when V. cholera lives outside a human host. When bacteria from contaminated drinking water encounter the environment of the human gut, this sets off a signal that activates an activator protein. The activator induces a response by binding to a regulatory DNA sequence near the promoters of all ToxR regulon genes to stimulate their transcription. The diarrhea induced by this regulon is adaptive for V. cholera because it spreads more bacteria into the environment to infect new hosts. What are the general messages of this chapter? Interactions among protein regulators and the DNA sequences they bind produce finely tuned control over gene expression, regulating individual genes, operons, or large sets of genes. With these exquisite controls over gene expression, bacteria have been able to compete, grow, and reproduce for more than 3 billion years of life s history. PROCESS: REGULON CONTROL OF GENE EXPRESSION Repressor Transcription blocked Operator BACTERIAL GENOME 1. Uninduced state: Genes of the regulon silenced by a common repressor. Regulon genes Inducing signal Repressor removed from operator Regulon products 2. Induced state: Repressor inactivated; regulon genes released for transcription and translation. RNA polymerase mrna Figure 11 Genes of a Regulon Are Expressed Together. The symbols indicate regions of the bacterial genome not shown between regulon genes. 443 Pg. No. 11

5 CHAPTER REVIEW For media, go to MasteringBiology If you understand... 1 You should be able explain how the operator, repressor, and inducer relate to a car s parking brake. An Overview of Gene Regulation and Information Flow Changes in gene expression allow bacterial cells to respond to environmental changes. 4 Positive Control of Transcription Most gene products are produced or activated only when they are needed. Positive control of transcription occurs when a regulatory protein called an activator binds to a regulatory sequence in DNA. Gene expression can be controlled at three levels: transcription, translation, or post-translation (protein activation). Activator proteins bind to RNA polymerase in addition to DNA. Binding between the activator and RNA polymerase increases the rate of transcription initiation. Transcriptional control can be negative or positive. Negative control occurs when a regulatory protein prevents transcription. Positive control occurs when a regulatory protein increases the frequency of initiating transcription. You should be able to describe one component of the lac operon that is under transcriptional control and one component that is under post-translational control. 2 You should be able to predict if mutations in the ara initiator sequence of the ara operon are most likely to affect positive regulation, negative regulation, or both. Identifying Regulated Genes Replica plating is a technique that allows researchers to identify mutants that cannot grow in a particular condition. Replica plating led to the isolation of three types of lactose metabolism mutants. Transcription may be constitutive or regulated. Constitutive expression occurs in genes whose products are required at all times, such as genes that encode glycolytic enzymes. You should be able to propose a strategy to isolate E. coli mutants that can grow at 33 C, but not at 42 C. 3 The ara operon codes for genes required for metabolism of the sugar arabinose. The operon is controlled by the AraC regulatory protein. AraC is an activator when bound to arabinose and a repressor when the protein is not bound to arabinose. 5 Global Gene Regulation Bacterial cells often need to coordinate the expression of large sets of genes in response to changing environments. Regulons coordinate the expression of different genes by using a shared regulator that acts on a regulatory sequence found in all genes of the regulon. Regulons can work through negative control using repressors, or through positive control using activators. You should be able to propose a method that would allow more genes to become part of the SOS regulon. Negative Control of Transcription Masteringbiology The lac operon is transcribed efficiently when lactose is present and glucose is absent. 1. Masteringbiology Assignments The lac operon is under negative control. Tutorials and Activities The lac Operon Negative control occurs because a repressor protein binds to an operator sequence in DNA near the promoter of the proteinencoding genes to prevent their transcription. Questions Reading Quizzes, Blue-Thread Questions, Test Bank When lactose is present, it binds to the repressor and causes it to fall off the operator, allowing transcription to occur. 3. The Study Area Practice Test, Cumulative Test, BioFlix 2. etext Read your book online, search, take notes, highlight text, and more. 3-D animations, Videos, Activities, Audio Glossary, Word Study Tools, Art Glucose inhibits transcription of the lac operon by inhibiting lactose transport into the cell. You should be able to... TEST YOUR KNOWLEDGE 1. Replica plating is used to isolate mutants that a. can produce an enzyme. b. cannot grow in a particular condition. 444 c. can utilize lactose. d. turn yellow when lactose is broken down. TEST YOUR UNDERSTANDING Pg. No. 12

6 2. Why are the genes involved in lactose metabolism considered to be an operon? a. They occupy adjacent locations on the E. coli chromosome. b. They have a similar function. c. They are all required for normal cell function. d. They are all controlled by the same promoter. 3. In the lac operon, the repressor inhibits transcription when a. the repressor is bound to the inducer. b. the repressor is not bound to the inducer. c. the repressor is bound to glucose. d. the repressor is not bound to the operator. 4. Activators bind to regulatory sequences in and to polymerase. 5. How does inducer exclusion control gene expression in the lac operon? 6. A regulon is a set of genes controlled by a. one type of regulator of transcription. b. two or more different alternative sigma proteins. c. many different types of promoters. d. glucose. TEST YOUR UNDERSTANDING 7. E. coli expresses genes for glycolytic enzymes constitutively. Why? 8. Explain the difference between positive and negative control over transcription. 9. Predict what would happen if the lac repressor protein were altered so it could not bind inducer. a. The repressor could not bind to DNA. b. The repressor would always be bound to DNA. c. The repressor could bind to DNA only when cells were grown with glucose. d. The repressor could bind to DNA only when cells were grown without glucose. 10. Predict what would happen to regulation of the lac operon if the laci gene were moved 50,000 nucleotides upstream of its normal location. 11. If any of the following hypothetical drugs could be developed, which would be most effective in preventing cholera? a. a drug that increased the amount of the ToxR activator b. a drug that blocked the DNA-binding activity of the activator c. a drug that increased rates of transcription in V. cholerae d. a drug that increased rates of translation in V. cholerae 12. IPTG is a molecule with a structure very similar to lactose. IPTG can be transported into cells by galactoside permease and can bind to the lac repressor protein. However, unlike lactose, IPTG is not broken down by β-galactosidase. Predict what would occur regarding lac operon regulation if IPTG were added to E. coli growth medium containing arabinose and no glucose or lactose. TEST YOUR PROBLEM-SOLVING SKILLS 13. You are interested in using bacteria to metabolize wastes at an old chemical plant and convert them into harmless compounds. You find bacteria that are able to tolerate high levels of the toxic compounds toluene and benzene, and you suspect that this is because the bacteria can break down these compounds into lesstoxic products. If that is true, these toluene- and benzene-resistant strains will be valuable for cleaning up toxic sites. How could you find out whether these bacteria are metabolizing toluene as a source of carbon compounds? 14. QuANTiTATiVe Imagine that you are repeating the replica-plating procedure of Jacob and Monod to find mutants that can t grow using lactose. After treating cells with a mutagen, you anticipate a mutation rate of lactose-nonutilizing mutants per mutagen-treated cell. Based on this estimate, how many cells should you replica-plate to have a good chance of finding one mutant? 15. A type of mutation in the lac operator known as laco c prevents repressor binding to DNA and causes constitutive transcription of the lac operon. Which of the following secondary mutations might restore normal regulation to the lac operon in a laco c mutant? a. a laci mutation that decreases the ability of the repressor to bind the inducer b. a laci mutation that produces a repressor than can recognize the mutated laco c DNA sequence c. a promoter mutation that prevents it from being recognized by sigma d. an RNA polymerase mutation that allows it to bind to the promoter without using sigma 16. X-gal is a colorless, lactose-like molecule that can be split into two fragments by β-galactosidase. One of these product molecules is blue. The following photograph shows E. coli colonies growing in a medium that contains X-gal. Find three colonies whose cells have functioning copies of β-galactosidase. Find three colonies whose cells might have mutations in the lacz or in the lacy genes. Suppose you analyze the protein-coding sequence of the lacz and lacy genes of cells from the three mutant colonies and find that these sequences are wild type (normal). What other region of the lac operon might be altered to account for the mutant phenotype of these colonies? 445

7 Answers Answers are listed in order of appearance. in-text QueSTiONS AND exercises See TAbLe A1. Write Slowest response, most efficient Fig. 1 resource use next to the transcriptional control label. Write Fastest response, least efficient resource use next to the post-translational control label. Fig. 2 Plates from all three treatments must be identical and contain identical growth medium, except for the presence of the sugars labeled in the figure. Also, all plates must be grown under the same physical conditions (temperature, light) for the same time. Fig. 3 Use a medium with all 20 amino acids when producing a master plate of mutagenized E. coli colonies; then use a replica plate that contains all the amino acids except tryptophan. Choose cells from the master plate that did not grow on the replica plate. lacz codes for the β-galactosidase enzyme, which breaks the disaccharide lactose into glucose and galactose. lacy codes for the galactoside permease enzyme, which transports lactose into the bacterial cell. laci codes for a protein that shuts down production of the other lac products. When lactose is absent, the laci product prevents transcription. This is logical because there is no reason for the cell to make β-galactosidase and galactoside permease if there is no lactose to metabolize. But when lactose is present, it interacts with laci in some way so that lacz and lacy are induced (their transcription can occur). When lactose is present, the enzymes that metabolize it are expressed. A mutation that prevents lactose binding to repressor is predicted to prevent transcription of the lac operon under any condition. This is because the repressor would never come off the operator. A mutation in the operator that prevents repressor binding is predicted to lead to constitutive expression of the lac operon. Fig. 8 Put the Repressor protein on the operator. No transcription will take place. Then put the RNA polymerase on the promoter. No transcription will take place. Finally, put lactose on the repressor protein and then remove the resulting lactose repressor complex from the operon. Transcription will begin. CYu (1) It is logical that the genes for metabolizing lactose should be expressed only when lactose is See Figure A1. available. (2) This mutation is predicted to lower rates of transcription initiation because AraC s binding to RNA polymerase is essential for AraC to work as an activator. YOu SHOuLD be AbLe TO... Test Your Knowledge 1. b 2. d 3. b 4. DNA; RNA; When glucose and another sugar are present 5. in the environment, inducer exclusion prevents the use of the other sugar and allows only use of glucose. 6. a Test Your understanding 7. The glycolytic enzymes are always needed in the cell because they are required to produce ATP, and ATP is always needed. 8. Positive control means that a regulatory protein, when present, causes transcription to increase. Negative control means that a regulatory protein, when present, prevents transcription. 9. b. 10. Regulation of the lac operon should be normal. The location of the laci gene isn t important, because the gene produces a protein that diffuses within the cell to the operator. 11. b (since the activator needs to bind to regulatory sequences to activate gene expression, preventing DNA binding would cripple the regulon and prevent cholera).12. The lac operon would be strongly induced. Once inside the cell, the IPTG will bind to the repressor, causing it to release from DNA. IPTG cannot be broken down, so its concentration will remain high. Finally, since glucose is absent, there will be no inducer exclusion to inhibit IPTG transport through the galactoside permease transporter. Test Your Problem-Solving Skills 13. Set up cultures with individuals that all come from the same colony of toluene-tolerating bacteria. Half the cultures should have toluene as the only source of carbon; half should have glucose or another common source of carbon. The glucose-containing medium serves as a control to ensure that cells can be grown in the lab. Cells will grow in both cultures if they are able to use toluene as a source of carbon; they will grow only in glucose-containing medium if toluene cannot be used as a carbon source. 14. At a rate of 1 * 10-4 mutants per cell, you would on average find one mutant in every 10,000 (1 * 104) cells. Therefore, you should screen a bit more (~ 2 3 times more) than 10,000 cells to be reasonably sure of finding at least one mutant. 15. b Cells with functioning β-galactosidase will 16. produce blue colonies; cells with lacz mutations or lacy mutations will not produce β-galactosidase and will produce white colonies. The lac promoter could be mutated so that RNA polymerase cannot bind. TAbLe A1 Figure A1 if YOu understand... 1 Production of β-galactosidase and galactosidase permease are under transcriptional control transcription depends on the action of regulatory proteins. The activity of the repressor is under post-translational control. 2 Treat E. coli cells with a mutagen, and create a master plate that is grown at 33 C. Replica-plate this master plate and grow the replica plate at 42 C. Look for colonies that are on the master plate at 33 C but not on the replica plate at 42 C. 3 The operator is the parking brake; the repressor locks it in place, and the inducer releases it. 4 ara initiator mutations are likely to affect positive and negative control because AraC must bind to the ara initiator sequence for both forms of control. 5 Mutations that create operators for the SOS regulon repressor protein would put new genes under control of the repressor and incorporate them into the regulon. 446 Pg. No. 14