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Chapter 14 Phage Strategies. 14.1 Introduction Bacteriophages are viruses that infect bacteria. Figure 14.CO: TEM of a T4 phage. © Dr. Harold Fisher/Visuals.

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Presentation on theme: "Chapter 14 Phage Strategies. 14.1 Introduction Bacteriophages are viruses that infect bacteria. Figure 14.CO: TEM of a T4 phage. © Dr. Harold Fisher/Visuals."— Presentation transcript:

1 Chapter 14 Phage Strategies

2 14.1 Introduction Bacteriophages are viruses that infect bacteria. Figure 14.CO: TEM of a T4 phage. © Dr. Harold Fisher/Visuals Unlimited

3 14.2 Lytic Development Is Divided into Two Periods A phage infective cycle is divided into the: –early period (before replication) –late period (after the onset of replication) A phage infection generates a pool of progeny phage genomes that replicate and recombine.

4 14.2 Lytic Development Is Divided into Two Periods Figure 14.1: A temperate phage may follow the lytic or lysogenic pathway.

5 14.2 Lytic Development Is Divided into Two Periods Figure 14.2: Phages reproduce in lytic development.

6 14.3 Lytic Development Is Controlled by a Cascade The early genes transcribed by host RNA polymerase following infection include, or comprise, regulators required for expression of the middle set of phage genes. The middle group of genes includes regulators to transcribe the late genes. This results in the ordered expression of groups of genes during phage infection.

7 14.3 Lytic Development Is Controlled by a Cascade Figure 14.3: Lytic development is a regulatory cascade.

8 14.4 Two Types of Regulatory Event Control the Lytic Cascade Regulator proteins used in phage cascades may: sponsor initiation at new (phage) promoters cause the host polymerase to read through transcription terminators

9 14.4 Two Types of Regulatory Event Control the Lytic Cascade Figure 14.4: A phage may control transcription at initiation either by synthesizing a new sigma factor that replaces the host sigma factor or by synthesizing a new RNA polymerase.

10 14.4 Two Types of Regulatory Event Control the Lytic Cascade Figure 14.5: Control at initiation generates independent mRNAs.

11 14.4 Two Types of Regulatory Event Control the Lytic Cascade Figure 14.6: Control at termination generates a single mRNA.

12 14.5Lambda Immediate Early and Delayed Early Genes Are Needed for Both Lysogeny and the Lytic Cycle Lambda has two immediate early genes, N and cro, which are transcribed by host RNA polymerase. N is required to express the delayed early genes.

13 Three of the delayed early genes are regulators. Lysogeny requires the delayed early genes cII- cIII. The lytic cycle requires the immediate early gene cro and the delayed early gene Q. 14.5Lambda Immediate Early and Delayed Early Genes Are Needed for Both Lysogeny and the Lytic Cycle

14 Figure 14.7: Lambda has two lifestyles.

15 14.6 The Lytic Cycle Depends on Antitermination by pN pN is an antitermination factor. –It allows RNA polymerase to continue transcription past the ends of the two immediate early genes. pQ is the product of a delayed early gene. –It is an antiterminator that allows RNA polymerase to transcribe the late genes.

16 14.6 The Lytic Cycle Depends on Antitermination by pN Figure 14.8: Lambda genes are functionally clustered.

17 14.6 The Lytic Cycle Depends on Antitermination by pN Figure 14.9: Similar controls apply to left and right transcription.

18 14.6 The Lytic Cycle Depends on Antitermination by pN Lambda DNA circularizes after infection. –The late genes form a single transcription unit. Figure 14.10: Lambda has three stages of development.

19 14.7 Lysogeny Is Maintained by the Lambda Repressor Protein The lambda repressor, encoded by the cI gene, is required to maintain lysogeny. The lambda repressor acts at the O L and O R operators to block transcription of the immediate early genes. The immediate early genes trigger a regulatory cascade. –Their repression prevents the lytic cycle from proceeding.

20 14.7 Lysogeny Is Maintained by the Lambda Repressor Protein Figure 14.11: Lambda has a compact regulatory region.

21 14.7 Lysogeny Is Maintained by the Lambda Repressor Protein Figure 14.12: Repressor maintains lysogeny

22 14.8 The Lambda Repressor and Its Operators Define the Immunity Region Several lambdoid phages have different immunity regions. A lysogenic phage confers immunity to further infection by any other phage with the same immunity region.

23 14.8 The Lambda Repressor and Its Operators Define the Immunity Region Figure 14.13: RNA polymerase initiates at P L and P R but not at P RM during the lytic cycle.

24 14.9 The DNA-Binding Form of the Lambda Repressor Is a Dimer A repressor monomer has two distinct domains. The N-terminal domain contains the DNA- binding site. The C-terminal domain dimerizes.

25 14.9 The DNA-Binding Form of the Lambda Repressor Is a Dimer Figure 14.14: Repressor has two domains.

26 14.9 The DNA-Binding Form of the Lambda Repressor Is a Dimer Binding to the operator requires the dimeric form so that two DNA-binding domains can contact the operator simultaneously. Cleavage of the repressor between the two domains: –reduces the affinity for the operator –induces a lytic cycle

27 14.9 The DNA-Binding Form of the Lambda Repressor Is a Dimer Figure 14.15: Repressor cleavage induces lytic cycle.

28 14.10 Repressor Uses a Helix-Turn-Helix Motif to Bind DNA Each DNA-binding region in the repressor contacts a half-site in the DNA. The DNA-binding site of the repressor includes two short α-helical regions that fit into the successive turns of the major groove of DNA.

29 14.10 Repressor Uses a Helix-Turn-Helix Motif to Bind DNA Figure 14.16: The operator is a 17-bp sequence with an axis of symmetry through the central base pair. Each half-site is marked in light blue. Base pairs that are identical in each operator half are in dark blue.

30 14.10 Repressor Uses a Helix-Turn-Helix Motif to Bind DNA Figure 14.18: Repressor binds DNA via two α-helices.

31 14.10 Repressor Uses a Helix-Turn-Helix Motif to Bind DNA A DNA-binding site is a (partially) palindromic sequence of 17 bp. The amino acid sequence of the recognition helix makes contacts with particular bases in the operator sequence that it recognizes.

32 14.10 Repressor Uses a Helix-Turn-Helix Motif to Bind DNA Figure 14.19: Helix-3 determines DNA-binding specificity.

33 14.11 Repressor Dimers Bind Cooperatively to the Operator Repressor binding to one operator increases the affinity for binding a second repressor dimer to the adjacent operator. The affinity is 10x greater for O L 1 and O R 1 than other operators, so they are bound first. Cooperativity allows repressor to bind the O L 1/O R 2 sites at lower concentrations.

34 14.11 Repressor Dimers Bind Cooperatively to the Operator Figure 14.21: Each operator contains three repressor-binding sites and overlaps with the promoter at which RNA polymerase binds..

35 14.11 Repressor Dimers Bind Cooperatively to the Operator Figure 14.22: Lambda repressors bind DNA cooperatively.

36 14.12 Lambda Repressor Maintains an Autoregulatory Circuit The DNA-binding region of repressor at O R 2 contacts RNA polymerase and stabilizes its binding to PRM. This is the basis for the autoregulatory control of repressor maintenance. Repressor binding at O L blocks transcription of gene N from P L.

37 14.12 Lambda Repressor Maintains an Autoregulatory Circuit Figure 14.23: Helix-2 interacts with RNA polymerase.

38 Repressor binding at O R blocks transcription of Cro, but also is required for transcription of cI. Repressor binding to the operators therefore simultaneously blocks entry to the lytic cycle and promotes its own synthesis Lambda Repressor Maintains an Autoregulatory Circuit

39 14.13 Cooperative Interactions Increase the Sensitivity of Regulation Repressor dimers bound at O L 1 and O L 2 interact with dimers bound at O R 1 and O R 2 to form octamers. These cooperative interactions increase the sensitivity of regulation.

40 14.13 Cooperative Interactions Increase the Sensitivity of Regulation Figure 14.24: In the lysogenic state, the repressors bound at O L 1 and O L 2 interact with those bound at O R 1 and O R 2. RNA polymerase in bound at P RM (which overlaps with O R 3) and interacts with the repressor bound at O R 2.

41 14.13 Cooperative Interactions Increase the Sensitivity of Regulation Figure 14.25: O L 3 AND O R 3 are brought into proximity by formation of the repressor octamer, and an increase in repressor concentration allows dimers to bind at these sites and interact.

42 14.14 The cII and cIII Genes Are Needed to Establish Lysogeny The delayed early gene products cII and cIII are necessary for RNA polymerase to initiate transcription at the promoter P RE. cII acts directly at the promoter and cIII protects cII from degradation. Transcription from P RE leads to synthesis of repressor and also blocks the transcription of cro.

43 14.14 The cII and cIII Genes Are Needed to Establish Lysogeny Figure 14.26: Repressor establishment uses a special promoter.

44 P RE has atypical sequences at –10 and –35. RNA polymerase binds the promoter only in the presence of cII. cII binds to sequences close to the –35 region The cII and cIII Genes Are Needed to Establish Lysogeny

45 14.15 Lysogeny Requires Several Events cII and cIII cause repressor synthesis to be established. –They trigger inhibition of late gene transcription. Establishment of repressor turns off immediate and delayed early gene expression. Repressor turns on the maintenance circuit for its own synthesis. Lambda DNA is integrated into the bacterial genome at the final stage in establishing lysogeny.

46 14.15 Lysogeny Requires Several Events Figure 14.27: The lysogenic pathway leads to repressor synthesis.

47 14.16 The Cro Repressor Is Needed for Lytic Infection Cro binds to the same operators as the lambda repressor, but with different affinities. When Cro binds to OR3, it: –prevents RNA polymerase from binding to PRM –blocks the maintenance of repressor promoter When Cro binds to other operators at O R or O L, it prevents RNA polymerase from expressing immediate early genes. –This (indirectly) blocks repressor establishment.

48 14.16 The Cro Repressor Is Needed for Lytic Infection Figure 14.28: The lytic pathway leads to expression of cro and late genes.

49 14.17 What Determines the Balance Between Lysogeny and the Lytic Cycle? The delayed early stage when both Cro and repressor are being expressed is common to lysogeny and the lytic cycle. The critical event is whether cII causes sufficient synthesis of repressor to overcome the action of Cro.

50 14.17 What Determines the Balance Between Lysogeny and the Lytic Cycle? Figure 14.29: Repressor determines lysogeny, and Cro determines the lytic cycle.


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