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Peter J. Russell A molecular Approach 2nd Edition CHAPTER 19 Regulation of Gene Expression in Bacteria and Bacteriophages edited by Yue-Wen Wang Ph. D. Dept. of Agronomy,台大農藝系 NTU 遺傳學 601 20000 Chapter 19 slide 1 The lac Operon of E. coli 1. Growth and division genes of bacteria are regulated genes. Their expression is controlled by the needs of the cell as it responds to its environment with the goal of increasing in mass and dividing. 2. Genes that generally are continuously expressed are constitutive genes (housekeeping genes). Examples include protein synthesis and glucose metabolism. 3. All genes are regulated at some level, so that as resources dwindle the cell can respond with a different molecular strategy. 4. Prokaryotic genes are often organized into operons that are cotranscribed. A regulatory protein binds an operator sequence in the DNA adjacent to the gene array, and controls production of the polycis-tronic (polygenic) mRNA. 5. Gene regulation in bacteria and phage is similar in many ways to the emerging information about gene regulation in eukaryotes, including humans. Much remains to be discovered; even in E. coli, one of the most closely studied organisms on earth, 35% of the genomic ORFs have no attributed function. 台大農藝系 遺傳學 601 20000 Chapter 19 slide 2 The lac Operon of E. coli Animation: Regulation of Expression of the lac Operon Genes 1. An inducible operon responds to an inducer substance (e.g., lactose). An inducer is a small molecule that joins with a regulatory protein to control transcription of the operon. 2. The regulatory event typically occurs at a specific DNA sequence (controlling site) near the protein-coding sequence (Figure 19.1). 3. Control of lactose metabolism in E. coli is an example of an inducible operon. 台大農藝系 遺傳學 601 20000 Chapter 19 slide 3 Fig. 19.1 General organization of an inducible gene Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings. 台大農藝系 遺傳學 601 20000 Chapter 19 slide 4 Lactose as a Carbon Source for E. coli 1. E. coli expresses genes for glucose metabolism constitutively, but the genes for metabolizing other sugars are regulated in a “sugar specific” sort of way. Presence of the sugar stimulates synthesis of the proteins needed. 2. Lactose is a disaccharide (glucose 1 galactose). If lactose is E. coli’s sole carbon source, three genes are expressed: a. β-galactosidase has two functions: i. Breaking lactose into glucose and galactose. Galactose is converted to glucose, and glucose is metabolized by constitutively produced enzymes. ii. Converting lactose to allolactose (an isomerization). Allolactose is involved in regulation of the lac operon (Figure 19.2). b. Lactose permease (M protein) is required for transport of lactose across the cytoplasmic membrane. c. Transacetylase is poorly understood. 3. The lac operon shows coordinate induction: a. In glucose medium, E. coli normally has very low levels of the lac gene products. b. When lactose is the sole carbon source, levels of the three enzymes increase coordinately (simultaneously) about 1,000-fold. i. Allolactose is the inducer molecule (Figure 16.2). ii. The mRNA for the enzymes has a short half-life. When lactose is gone, lac transcription stops, and enzyme levels drop rapidly. 台大農藝系 遺傳學 601 20000 Chapter 19 slide 5 Fig. 19.2 Reactions catalyzed by the enzyme -galactosidase Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings. 台大農藝系 遺傳學 601 20000 Chapter 19 slide 6 Experimental Evidence for the Regulation of lac Genes 1. The experiments of Jacob and Monod produced an understanding of arrangement and control of the lac genes. 台大農藝系 遺傳學 601 20000 Chapter 19 slide 7 Mutations in the Protein-coding Genes 1. Mutagens produced mutations in the lac structural genes that were used to map their locations. a. β-galactosidase is lacZ. b. Permease is lacY. c. Transacetylase is lacA. d. The genes are tightly linked in the order: lacZ-lacY-lacA 2. The type of mutation made a difference in expression of the downstream genes: a. Missense mutations affect only the product of the gene with the mutation. b. Nonsense mutations show polarity (polar mutations), and affect translation of the downstream genes as well. 3. The interpretation of gene polarity is that ribosomes translate the first gene in the polycistronic (polygenic) RNA, and finish in proper position to initiate and translate the next gene. Premature translation termination prevents this by reducing translation of the downstream genes (Figure 19.3). 台大農藝系 遺傳學 601 20000 Chapter 19 slide 8 Fig. 19.3 Translation of the polygenic mRNA encoded by lac utilization genes Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings. 台大農藝系 遺傳學 601 20000 Chapter 19 slide 9 Mutations Affecting the Regulation of Gene Expression 1. Jacob and Monod found mutants that produced all three lac operon proteins constitutively, and hypothesized that regulatory mutations had affected the normal control of gene expression for the operon. Constitutive mutants are in two classes (Figure 19.4): a. Mutations in the lac operator (lacO) just upstream from the lacZ gene. b. Mutations further upstream in the lac repressor gene (lacI). 2. Operator-constitutive (lacOc) mutations were defined by experiments using partial diploid E. coli F’ strains. a. An example is the partial diploid F’ lacO+ lacZ- lacY+/lacOc lacZ+ lacY-. (Promoters are normal for both operons, and the lacA gene is irrelevant to the study.) b. This strain was tested for β-galactosidase and lactose permease, both in the presence and absence of the inducer. c. Without inducer, β-galactosidase is produced, but only inactive permease is made. i. β-galactosidase is produced from the chromosomal gene under control of the constitutive promoter. ii. Permease is produced from the chromosomal gene also, but is inactive because the gene is mutated. iii. No products are produced from the F’ DNA, because its promoter is wild-type, and requires induction for gene expression. 台大農藝系 遺傳學 601 20000 Chapter 19 slide 10 Fig. 19.4 Organization of the lac genes of E. coli and the associated regulatory elements: the lac operon Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings. 台大農藝系 遺傳學 601 20000 Chapter 19 slide 11 d. With inducer, functional molecules of both β-galactosidase and permease are produced. This indicates that: i. β-galactosidase is under constitutive control (on the chromosome). ii. Permease is under inducible control (on the F’ DNA). e. In both cases, the promoter controls genes downstream from it on the same DNA molecule, showing cis-dominance. 3. lacI constitutive genes were also discovered in experiments with partial diploid strains. a. An example is the strain lacI+ lacO+ lacZ- lacY+/lacI- lacO+ lacZ+ lacY-, in which both gene sets have normal operators and promoters. b. Without inducer, no β-galactosidase or permease is produced. c. With inducer, both are produced. A lacI+ gene can overcome the lacI- mutation. d. Since lacI+ and lacI- genes are on different DNA molecules, lacI+ is trans-dominant. e. Jacob and Monod proposed that lacI+ produces a repressor that controls expression of both lac operons, making them both inducible. 4. The lac promoter is also affected by mutations (Plac-). Most affect all three structural genes, which are not made, even when the inducer is present. a. Plac- mutations affect RNA polymerase binding to the start of the operon. b. Only genes in the same DNA strand are affected, so Plac- mutations are cis-ominant. iActivity: Mutations and Lactose Metabolism 台大農藝系 遺傳學 601 20000 Chapter 19 slide 12 Jacob and Monod’s Operon Model for the Regulation of lac Genes 1. Jacob and Monod’s model of regulation, with more recent information, follows: a. An operon is a cluster of genes that are regulated together. The order of the lac genes is shown in Figure 19.4, and Figure 19.5 shows the operon when lactose is absent. b. The lacI gene has its own constitutive weak promoter and terminator, and repressor protein is always present in low concentration. i. The repressor functions as a tetramer (Figure 19.6). ii. Repressor protein binds the operator (lacO+), and prevents RNA polymerase initiation to transcribe the operon genes (negative control). iii. Binding of the repressor to the operator is not absolute, and so an occasional transcript is made, resulting in low levels of the structural proteins. c. β-galactosidase in wild-type E. coli growing with lactose as the sole carbon source converts lactose into allolactose (Figures 19.2 and 19.7). i. Repressor bound with allolactose bound changes shape (allosteric shift) and dissociates from the lac operator. Free repressor-allolactose complexes are unable to bind the operator. ii. Allolactose induces expression of the lac operon, by removing the repressor and allowing transcription to occur. 台大農藝系 遺傳學 601 20000 Chapter 19 slide 13 Fig. 19.5 Functional state of the lac operon in wild-type E. coli growing in the absence of lactose Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings. 台大農藝系 遺傳學 601 20000 Chapter 19 slide 14 Fig. 19.6 Molecular model of the lac repressor tetramer Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings. 台大農藝系 遺傳學 601 20000 Chapter 19 slide 15 Fig. 19.7 Functional state of the lac operon in wild-type E. coli growing in the presence of lactose as the sole carbon source 台大農藝系 遺傳學 601 20000 Chapter 19 slide 16 2. The lacOc mutations result in constitutive gene expression. They are cis-dominant to lacO+, because repressor cannot bind to the lacOc operator sequence (Figure 19.8). 3. The lacI- mutations change the repressor protein’s conformation and prevent it from binding the operator, resulting in constitutive expression of the operon (Figure 19.9). a. In a partial diploid (lacI+ lacO+ lacZ- lacY+/lacI- lacO+ lacZ+ lacY-), the wild-type repressor (lacI+) is dominant over lacI- mutants. b. Defective lacI- repressor can’t bind either operator, but normal repressor from lacI+ binds both operators and regulates transcription, resulting in functional β-galactosidase and permease. 台大農藝系 遺傳學 601 20000 Chapter 19 slide 17 Fig. 19.8a Cis-dominant effect of lacOc mutation in a partial-diploid strain of E. coli 台大農藝系 遺傳學 601 20000 Chapter 19 slide 18 Fig. 19.8b Cis-dominant effect of lacOc mutation in a partial-diploid strain of E. coli 台大農藝系 遺傳學 601 20000 Chapter 19 slide 19 Fig. 19.9a Effects of a lacI- mutation 台大農藝系 遺傳學 601 20000 Chapter 19 slide 20 Fig. 19.9b Effects of a lacI- mutation 台大農藝系 遺傳學 601 20000 Chapter 19 slide 21 Fig. 19.9c Effects of a lacI- mutation 台大農藝系 遺傳學 601 20000 Chapter 19 slide 22 4. Additional lacI mutants have been identified: a. Superrepressor (lacIS) mutants that produce no lac enzymes. i. The mutant repressor cannot bind allolactose, so lactose does not induce the operon. ii. The lacIS allele is trans-dominant in partial diploids (lacI+/lacIS) (Figure 19.10). The superrepressor protein binds both operators and transcription cannot occur. iii. Normal repressor cannot compete, because superrepressor cannot be induced to fall off. iv. Low levels of transcription will occur (superrepressor is not covalently bound to the DNA operator sequence), but lacIS E. coli cannot use lactose as a carbon source. b. Dominance (lac-d) mutants have missense mutations at the 5’ end of the lacI gene. i. In haploid cells, the phenotype is constitutive expression of the lac operon. ii. In partial diploids, lac-d is trans-dominant. The operon is expressed even in the presence of normal repressor. iii. Normal repressor functions as a tetramer. Mutant and normal subunits combine randomly. iv. A tetramer containing one or more mutant subunits cannot bind to operator DNA. Repression does not occur, and so gene expression is constitutive. c. Mutations in the lad promoter can increase or decrease the gene¡¦s transcription rate, by altering its affinity for RNA polymerase. Examples are lacIQ and lacSQ: i. Both of these mutations raise the transcription rate of the repressor gene. (Q stands for “quantity” and SQ for “super quantity”) ii. Large amounts of repressor are produced in these mutants, reducing the efficiency of lac operon induction so that high levels of lactose are needed. lii. Overproduction of the lad gene has been useful for isolating and characterizing the repressor molecule. 5. These mutants indicate that repressor has three different recognition interactions: a. Binding to the operator region. b. Binding with the inducer (allolactose). c. Binding of repressor polypeptide subunits to form an active tetramer. 台大農藝系 遺傳學 601 20000 Chapter 19 slide 23 Fig. 19.10 Dominant effect of lacIs mutation over wild-type lacI+ 台大農藝系 遺傳學 601 20000 Chapter 19 slide 24 Positive Control of the lac Operon Animation: Positive Control of the lac Operon 1. Repressor exerts negative control by preventing transcription. Positive control of this operon also occurs when lactose is E. coli’s sole carbon source (Figure 19.11). a. Catabolite activator protein (CAP) binds cyclic AMP (cAMP) (Figure 19.12). b. CAP-cAMP complex is a positive regulator of the lac operon. It binds the CAP-site, a DNA sequence upstream of the operon’s promoter. c. Binding of CAP-cAMP complex causes the DNA to bend, facilitating protein-protein interactions between CAP and RNA polymerase, and leading to transcription. 2. When both glucose and lactose are in the medium, E. coli preferentially uses glucose, due to catabolite repression. a. Glucose metabolism greatly reduces cAMP levels in the cell. b. The CAP-cAMP level drops, and is insufficient to maintain high transcription of the lac genes. c. Even when allolactose has removed the repressor protein from the operator, lac gene transcription is at very low levels without CAP-cAMP complex bound to the CAP-site. d. Experimental evidence supports this model. Adding cAMP to cells restored transcription of the lac operon, even when glucose was present. 台大農藝系 遺傳學 601 20000 Chapter 19 slide 25 Fig. 19.11 Role of cyclic AMP (cAMP) in the functioning of glucose-sensitive operons such as the lac operon of E. coli 台大農藝系 遺傳學 601 20000 Chapter 19 slide 26 Fig. 19.12 Structure of cyclic AMP (cAMP) 台大農藝系 遺傳學 601 20000 Chapter 19 slide 27 3. The model is that catabolite repression targets adenylate cyclase (the enzyme that makes cAMP) (Figure 19.12). a. In E. coli, the phosphorylated form of IIIGlc enzyme activates adenylate cyclase. b. Glucose transport into the cell triggers events including dephosphorylation of IIIGlc. c. With IIIGlc protein dephosphorylated, adenylate cyclase is inactivated, and no new cAMP is produced. 4. Catabolic genes for other sugars are also regulated by catabolite repression. In all cases, a CAP site in their promoters is bound by a CAP-cAMP complex, increasing RNA polymerase binding. 台大農藝系 遺傳學 601 20000 Chapter 19 slide 28 Molecular Details of lac Operon Regulation 1. The sequences of significant lac regulatory regions are known. DNase protection by regulatory molecules (e.g., repressor) is useful in these studies. 2. The lacI DNA sequence shows the expected transcription and translation signals, except that the start codon is GUG (not AUG). The single base-pair mutation of IacIQ is also characterized (Figure 19.13). 3. Operon controlling sites (Figure 19.14) were derived from several types of data: a. Amino acid sequences of the repressor protein and (β-galactosidase were known, allowing coding regions for lacI and lacZ+ to be identified. b. Protection assays identified binding sites for: i. CAP-cAMP complex. ii. RNA polymerase. iii. Repressor protein. 台大農藝系 遺傳學 601 20000 Chapter 19 slide 29 Fig. 19.13 Base pair sequences of the lac operon lacI+ gene promoter (Plac+) and of the 5' end of the repressor mRNA 台大農藝系 遺傳學 601 20000 Chapter 19 slide 30 Fig. 19.14 Base pair sequence of the controlling sites, promoter and operator, for the lactose operon of E. coli 台大農藝系 遺傳學 601 20000 Chapter 19 slide 31 4. The lac operon promoter region begins at -84, immediately next to the lacI gene stop codon, and ends at -8, just upstream from the transcription start site. Features of the promoter region include: a. The consensus sequence for CAP-cAMP binding is in two regions: -54 to -58, and 65 to -69. b. The RNA polymerase binding site (including a Pribnow box) spans DNA from -47 to -8, with consensus sequence matches at -10 and -35. 5. The operator is immediately next to the promoter, with repressor protein protecting DNA from -3 to +21. With repressor bound to the operator, RNA polymerase can bind but cannot transcribe. 6. The operon transcript begins at +1, which is within the operator region bound by repressor. (Part of the operator is transcribed.) a. The β-ga1actosidase gene has a leader region before the start codon. b. Start codon for 3-galactosidase (AUG) is at + 39 to +41. c. Several lacOC mutations have been characterized. All are single base pair substitutions. 7. The lac operon was the first molecular model for gene regulation. Operons are common in bacteria and phages, but unknown in eukaryotes. 台大農藝系 遺傳學 601 20000 Chapter 19 slide 32 The trp Operon of E. coli 1. If amino acids are available in the medium, E. coli will import them rather than make them, and the genes for amino acid biosynthesis are repressed. When amino acids are absent, the genes are expressed and biosynthesis occurs. 2. Unlike the inducible lac operon, the trp operon is repressible. Generally, anabolic pathways are repressed when the end product is available. 台大農藝系 遺傳學 601 20000 Chapter 19 slide 33 Gene Organization of the Tryptophan Biosynthesis Genes 1. Yanofsky and colleagues characterized the controlling sites and genes of the trp operon (Figure 19.15). a. There are 5 structural genes, trpA through E. b. The promoter and operator are upstream from the trpE gene. c. Between trpE and the promoter-operator is trpL, the leader region. Within trpL is the attenuator region (att). d. The trp operon spans about 7 kb. The operon produces a polygenic transcript with five structural genes for tryptophan biosynthesis. 台大農藝系 遺傳學 601 20000 Chapter 19 slide 34 Fig. 19.15 Organization of controlling sites and the structural genes of the E. coli trp operon Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings. 台大農藝系 遺傳學 601 20000 Chapter 19 slide 35 Regulation of the trp Operon Animation: Attenuation in the trp Operon of E. coli 1. Two mechanisms regulate expression of the trp operon: a. Repressor/operator interaction. b. Transcription termination. 2. When tryptophan is present, it will bind to an aporepressor protein (the trpR gene product). a. The active repressor (aporepressor plus tryptophan) binds the trp operator, and prevents transcription initiation. b. Repression reduces transcription of the trp operon about 70-fold. 3. When tryptophan is limited, transcription is also controlled by attenuation. a. Attenuation produces only short (140-bp) transcripts that do not encode structural proteins. b. Termination occurs at the attenuator site within the trpL region. c. The proportion of attenuated transcripts to full-length ones is related to tryptophan levels, with more attenuated transcripts as the tryptophan concentration increases. d. Attenuation can reduce trp operon transcription 8- to 10-fold. Together, repression 601 20000 and attenuation regulate trp gene expression台大農藝系 over a 560-遺傳學 to 700-fold range.Chapter 19 slide 36 4. The molecular model for attenuation: a. Translation of the trpL gene produces a short polypeptide. Near the stop codon are two tryptophan codons. b. Within the leader mRNA are four regions that can form secondary structures by complementary base-pairing (Figure 19.16). i. Pairing of sequences 1 and 2 creates a transcription pause signal. ii. Pairing of sequences 3 and 4 is a transcription termination signal. iii. Pairing of 2 and 3 is an antitermination signal, and so transcription will continue. c. Tight coupling of transcription and translation in prokaryotes makes control by attenuation possible. i. RNA polymerase pauses when regions 1 and 2 base pair just after they are synthesized (Figure 19.16). 台大農藝系 遺傳學 601 20000 Chapter 19 slide 37 ii. During the pause, a ribosome loads onto the mRNA and begins translation of the leader peptide. Ribosome position is key to attenuation: (1) When tryptophan (Trp) is scarce(Figure 19.17): (a) Trp-tRNAs are unavailable, and the ribosome stalls at the Trp codons in the leader sequence, covering attenuator region 1. (b) When the ribosome is stalled in attenuator region 1, it cannot base pair with region 2. Instead, region 2 pairs with region 3 when it is synthesized. (c) If region 3 is paired with region 2, it is unable to pair with region 4 when it is synthesized. Without the region 3-4 terminator, transcription continues through the structural genes. (2) When Trp is abundant: (a) The ribosome continues translating the leader peptide, ending in region 2. This prevents region 2 from pairing with region 3, leaving 3 available to pair with region 4. (b) Pairing of regions 3 and 4 creates a rho-independent terminator known as the attenuator. Transcription ends before the structural genes are reached. 5. Genetic evidence for attenuation includes: a. Mutations in the leader sequence within regions 3 or 4 disrupt base pairing and decrease the efficiency of termination (Figure 19.18) b. Changes in the Trp codons of the leader peptide sequence, so that they encode a different amino acid, cause attenuation controlled by levels 台大農藝系 遺傳學 601 20000 Chapter 19 slide 38 of the amino acid specified by the mutant codon, not by Trp Fig. 19.16 Four regions of the trp operon leader mRNA and the alternate secondary structures they can form by complementary base-pairing Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings. 台大農藝系 遺傳學 601 20000 Chapter 19 slide 39 Fig. 19.17a Models for attenuation in the trp operon of E.coli Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings. 台大農藝系 遺傳學 601 20000 Chapter 19 slide 40 Fig. 19.17b Models for attenuation in the trp operon of E.coli Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings. 台大農藝系 遺傳學 601 20000 Chapter 19 slide 41 Fig. 19.18 In the trpL region, mutation sites that show less efficient transcription at the attenuator site Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings. 台大農藝系 遺傳學 601 20000 Chapter 19 slide 42 Regulation of Other Amino Acid Biosynthesis Operons 1.Attenuation is involved in regulating many operons(Figure 19.19). a.When the operon is for amino acid biosynthesis, the leader sequence always includes codons for that amino acid. b.Other operons regulated by attenuation include rRNA (rrn) and E. coli ampicillin resistance (ampC). 台大農藝系 遺傳學 601 20000 Chapter 19 slide 43 Fig. 19.19 Predicted amino acid sequences of the leader peptides of a number of attenuator-controlled bacterial operons Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings. 台大農藝系 遺傳學 601 20000 Chapter 19 slide 44 Regulation of Gene Expression in Phage Lambda 1. Phage use many bacterial components for replication, and control their use with phage gene products. 2. Bacteriophage λ has two possible pathways when it enters its E. coli host: a. The lytic cycle, in which the phage takes over the cell and produces progeny phage. b. The lysogenic cycle, where phage chromosome is inserted into the E. coli chromosome, and replicates with the bacterial genome. 台大農藝系 遺傳學 601 20000 Chapter 19 slide 45 Fig. 19.20 A map of phage , showing the major genes Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings. 台大農藝系 遺傳學 601 20000 Chapter 19 slide 46 Early Transcription Events 1. The λ chromosome is linear, with “sticky” ends used to circularize it in the host cell. The regulatory system for choosing between the lytic and lysogenic pathways is contained in the λ chromosome (Figure 19.20). a. First, transcription begins at promoters PL (leftward transcription) and PR (rightward) (Figure 19.21). i. The first gene transcribed from PR is cro (control of repressor and other). The Cro protein is involved in the genetic switch to the lytic pathway. ii. The first protein transcribed from PL is N, which is a transcription antiterminator that allows RNA synthesis to go through termination regions into the early genes. (1) N protein allows expression of the cII protein, which in turn activates: (a)cI (λ repressor) (b)O and P (DNA replication proteins). (c) Q (activation of late genes for lysis and phage particle proteins, only when Q protein accumulates to certain levels). 台大農藝系 遺傳學 601 20000 Chapter 19 slide 47 Fig. 19.21 Expression of genes after infecting E. coli and the transcriptional events that occur when either the lysogenic or lytic pathways are followed Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings. 台大農藝系 遺傳學 601 20000 Chapter 19 slide 48 Fig. 19.21 Expression of genes after infecting E. coli and the transcriptional events that occur when either the lysogenic or lytic pathways are followed (cont.) Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings. 台大農藝系 遺傳學 601 20000 Chapter 19 slide 49 The Lysogenic Pathway 1. Early transcription events determine whether the lytic or lysogenic pathway occurs. 2. The lysogenic pathway results when cII and cIII are expressed. a. The action of cII and cIII proteins activates the PRE promoter, causing transcription of the cI (λ repressor) gene. b. λ repressor binds to 2 operator regions, OL and OR, whose sequences overlap PL and PR, respectively. This prevents transcription from PL and PR. c. Transcription of N and cro genes is blocked, and concentrations of their proteins drop rapidly. d. In addition, repressor bound to OR causes more repressor mRNA to be made from another promoter, PRM. e. High levels of repressor cause lysogeny by binding operators OL and OR. f. Integrase protein is used to integrate λ DNA into the E. coli chromosome. Integrase transcript is initiated at the PI promoter, which is controlled by the cII protein. g. As cII concentration drops, the PI promoter is shut down, leaving PRM as the only active promoter. 3. Thus, the lysogenic pathway occurs when enough λ repressor is made to turn off early promoters. Lytic genes, including Q, are not expressed. Without the Q protein, phage coat and lysis proteins are not produced. 台大農藝系 遺傳學 601 20000 Chapter 19 slide 50 The Lytic Pathway 1. An example of induction of the lytic pathway is exposure to UV light. a. UV causes the bacterial RecA protein (normally used in DNA repair) to stimulate the λ repressor proteins to autocleave and become inactivated. b. Absence of repressor at OR allows transcription of the cro gene. c. Cro protein decreases RNA synthesis from PL and PR, reducing synthesis of cII, therefore blocking synthesis of λ repressor. d. Transcription from PR is also decreased, but Q protein levels are sufficient to allow transcription of the late genes needed for the lytic pathway. 2. Thus, λ uses complex regulatory systems to control entry into the lytic or lysogenic pathway. The decision depends on competition between the repressor and the Cro protein. a. If repressor dominates, lysogeny takes place. b. If the Cro protein dominates, the lytic pathway occurs. 台大農藝系 遺傳學 601 20000 Chapter 19 slide 51