Chapter 8 Major Shifts in Prokaryotic Transcription

1. Chapter 8 Major Shifts in Prokaryotic Transcription

2. 8.1 Modification of The Host RNA Polymerase During Phage Infection • SPO1(B. subtilis phage, large DNA genome) Temporal program of transcription

3. Figure 8.1 Temporal control of transcription In phage SPO1- infected B. subtilis. (a) Early transcription is directed by the host RNA polymerase holoenzyme, including the host σ factor (blue); one of the early phage proteins is gp28 (green), a new σ factor. (b) Middle transcription is directed by gp28, in conjunction with the host core polymerase (red); two middle phage proteins are gp33 and gp34 (purple and yellow, respectively); together, these constitute yet another σ factor. (c) Late transcription depends on the host core polymerase plus gp33 and 34.

4. Evidence for σ switching model • Genetic studies mutations in gene 28 prevent early-to-middle switch; mutations in gene 33 or 34 prevent middle-to-late switch • Biochemical studies purification of RNA polymerase

5. Figure 8.2 Subunit compositions of RNA polymerases in SP01 phage-infected B. subtilis cells. Polymerases were separated by chromatography and subjected to SDS-PAGE to display their subunits. Enzyme B (first lane) contains the core subunits (β', β, and α), as well as subunit IV (gp28). Enzyme C (second lane) contains the core subunits plus subunits V (gp33) and Vl (gp34). The last two lanes contain separated δ and σ subunits, respectively.

6. Figure 8.3 Specificities of polymerases B and C. Pero et al. measured polymerase specificity by transcribing SP01 DNA in vitro with core polymerase (a), enzyme B (b), or enzyme C (c), in the presence of [3H]UTP to label the RNA product. Next they hybridized the labeled RNA to SP01 DNA in the presence of each of the following competitors: early SP01 RNA (green) made in vivo in the presence of chloramphenicol (CAM); middle RNA (blue) collected from phage-infected cells at 10 minutes post-infection; and late RNA (red) collected from phage-infected cells 30 minutes post- infection, The product of the core polymerase is competed roughly equally by all three classes of RNA. On the other hand, competition for the product made by B plus δ is clearly competed best by middle RNA, and the product made by C plus δ is competed best by late RNA. These differences are not as dramatic as one might prefer, but they are easiest to see at low competitor concentration.

7. SUMMARY Transcription of phage SPO1 genes in infected B. subtilis cells proceeds according to a temporal program in which early genes are transcribed first, then middle genes, and finally late genes. This switching is directed by a set of phage-encoded σ factors that associate with the host core RNA polymerase and change its specificity from early to middle to late. The host σ is specific for the phage early genes; the phage gp28 protein switches the specificity to the middle genes; and the phage gp33 and gp34 proteins switch to late specificity.

8. 8.2 The RNA Polymerase Encoded in Phage T7 • T7 (E. coli phage, small genome) Temporal control of transcription in T7

9. Figure 8.4 Temporal control of transcription in phage T7-infected E. coil. (a) Early (class I) transcription depends on the host RNA polymerase holoenzyme, including the host σ factor (blue); one of the early phage proteins is the T7 RNA polymerase (green). (b) Late (class II and III) transcription depends on the T7 RNA polymerase.

10. SUMMARY Phage T7, instead of coding for a new σfactor to change the host polymerase's specificity from early to late, encodes a new RNA polymerase with absolute specificity for the later phage genes. This polymerase, composed of a single polypeptide, is a product of one of the earliest phage genes, gene 1. The temporal program in the infection by this phage is simple. The host polymerase transcribes the earliest (class I) genes, one of whose products is the phage polymerase, which then transcribes the later (class II and class III) genes.

11. 8.3 Control of transcription During Sporulation Figure 8.5 Two types of B.subtilis cells. (a) B.subtilis vegatative cells and (b) a sporulating cell. With an endospore developing at the left end.

12. Figure 8.6 Map of part of plasmid p213. This DNA region contains two promoters: a vegetative promoter (Veg) and a sporulation promoter (0.4 kb). The former is located on a 3050 bp EcoRI-HincII fragment (blue); the latter is on a 770 bp fragment (red).

13. Figure 8.7 Specificities of σA and 6E. Losick and colleagues transcribed plasmid p213 in vitro with RNA polymerase containing σA (lane 1) or σE (lane 2). Next they hybridized the labeled transcripts to Southern blots containing EcoRI-Hincll fragments of the plasmid. As shown in Figure 8.6, this plasmid has a vegetative promoter in a 3050 bp EcoRI-Hincll fragment, and a sporulation promoter in a 770 bp fragment. Thus, transcripts of the vegetative gene hybridized to the 3050 bp fragment, while transcripts of the sporulation gene hybridized to the 770 bp fragment. The autoradiogram in the figure shows that the σA enzyme transcribed only the vegetative gene, while the σE enzyme transcribed both the vegetative and sporulation genes.

14. Figure 8.8 Specificity of σE determined by run-off transcription from the spollD promoter. Rong et al. prepared a restriction fragment containing the spollD promoter and transcribed it in vitro with B. subtilis core RNA polymerase plus σE (middle lane) or σB plus σc (right lane) Lane M contained marker DNA fragments whose sizes are indicated at left The arrow at the right indicates the position of the expected run-off transcript from the spollD promoter (about 700 nt). Only the enzyme containing σE made this transcript.

15. SUMMARY When the bacterium B. subtilis sporulates, a whole new set of sporulation-specific genes is turned on, and many, but not all, vegetative genes are turned off. This switch takes place largely at the transcription level. It is accomplished by several new σfactors that displace the vegetative σfactor from the core RNA polymerase and direct transcription of sporulation genes instead of vegetative genes. Each σfactor has its own preferred promoter sequence.

16. 8.4 Genes with Multiple Promoter • The B. subtilis spoVG Gene • The Anabaena Glutamine Synthetase Gene • The E. coli glnA Gene

17. Figure 8.10 Resolution of RNA polymerases that transcribe the spoVG gene from two different promoters.

18. Figure 8.10 Resolution of RNA polymerases that transcribe the spoVG gene from two different promoters. Losick and his colleagues purified polymerase from B. subtilis ceils that were running out of nutrients. The last purification step was DNA-cellutose column chromatography. The polymerase activity in each fraction from the column is given by the red line and the scale on the left-hand y axis. The salt concentration used to remove the enzyme from the column is given by the green line and the scale on the right-hand y-axis. The inset shows the results of a run-off transcription assay using a DNA fragment with two spoVG promoters spaced 10 bp apart, The fraction numbers at the top of the inset correspond to the fraction numbers from the column at bottom. The last lane (M) contained marker DNA fragments. The two arrowheads at the left of the inset indicate the two run-off transcripts, approximately 110 and 120 nt in length. The column separated a polymerase that transcribed selectively from the downstream promoter and produced the shorter run-off transcript (fractions 19 and 20) from a polymerase that transcribed selectively from the upstream promoter and produced the longer run-off transcript (fractions 22 and 23).

19. Figure 8.11 Specificities of σB and σE. LOSiCk and colleagues purified sigma factors σ B and σE by gel electrophoresis and tested them with core polymerase by the same run-off transcription assay used in Figure 8.10. Lane 2, containing σE, caused initiation selectively at the downstream promoter (P2). Lane 5, containing σB, caused initiation selectively at the upstream promoter (P1). Lane 6, containing both σ factors caused initiation at both promoters. The other lanes were the results of experiments with other fractions containing neither σ factor.

20. Figure 8.11 Overlapping promoters in B.subtills spoVG. P1 denotes the upstream promoter, recognized by σ B; the start of transcription and -10 and -35 boxes for this promoter are indicated in red above the sequence. P2 denotes the downstream promoter, recognized by σE; the start of transcription and -10 and -35 boxes for this promoter are indicated in blue below the sequence.

21. Summary Some prokaryotic genes must be transcribed under conditions where two different σ factors are active. These genes are equipped with two different promoters, each recognized by one of the two σ factors. This ensures their expression no matter which factor is present and allows for differential control under different conditions.

22. 8.5 The E. coli Heat Shock Genes • htpR gene, σ32 (σH) • Comparison of σ32 and σ70 gene: -35 sequence space -10 sequence σ70 TTGACA 16-18 TATAA σ32 CNTTGAA 13-15 CCCCATNT

23. SUMMARY The heat shock response in E. coli is governed by an alternative σ factor, σ32 (σH) which displaces σ70 (σA) and directs the RNA polymerase to the heat shock gene promoters. The accumulation of σ32 in response to high temperature is due to stabilization of σ32 and enhanced translation of the mRNA encoding σ32 .

24. 8.6 Infection of E. coli by Phage λ Phage lambda can replicate in either of two ways: lytic or lysogenic. In the lytic mode, almost all of the phage genes are transcribed and translated, and the phage DNA is replicated, leading to production of progeny phages and lysis of the host cells. In the lysogenic mode, the lambda DNA is incorporated into the host genome; after that occurs, only one gene is expressed. The product of this gene, the lambda repressor, prevents transcription of all the rest of the phage genes. However, the incorporated phage DNA (the prophage) still replicates, since it has become part of the host DNA.

25. Figure 8.12 Lytic versus lysogenic infection by phage λ. Blue cells are in the lytic phase; yellow cells are in the lysogenic phase; green cells are uncommitted.

26. Summary

27. Figure 8.13 Genetic map of phage lambda. (a) The map is shown in linear form, as the DNA exists in the phage particles; the cohesive ends (cos) are at the ends of the map. The genes are grouped primarily according to function. (b) The map is shown in circular form, as it exists in the host cell during a lyric infection after annealing of the cohesive ends.

28. Lytic Reproduction of λ Phage The immediate early/delayed early/late transcriptional switching in the lytic cycle of phage lambda is controlled by antiterminators. One of the two immediate early genes is cro, which codes for a repressor of the cI gene that allows the lytic cycle to continue. The other, N, codes for an antiterminator, N, that overrides the terminators after the N and cro genes. Transcription then continues into the delayed early genes. One of the delayed early genes, Q, codes for another antiterminator (Q) that permits transcription of the late genes from the late promoter, PR', to continue without premature termination.

29. Figure 8.14 Temporal control of transcription during lytic infection by phage lambda. (a) Immediate early transcription (red) starts at the rightward and leftward promoters (PR‘ and PL, respectively) that flank the repressor gene (cI); transcription stops at the rho-dependent terminators (t) after the N and cro genes. (b) Delayed early transcription (blue) begins at the same promoters, but bypasses the terminators by virtue of the N gene product. N. which is an antiterminator. (c) Late transcription (gray) begins at a new promoter (PR'); it would step short at the terminator (t) without the Q gene product, Q, another antiterminator. Note that O and P are protein- encoding delayed early genes, not operator and promoter.

30. Figure 8.15 Effect of N on leftward transcription. (a) Map of N region of λ genome. The genes surrounding N are depicted, along with the leftward promoter (PL) and operator (OL), the terminator (red), and the nut site (green). (b) Transcription in the absence of N. RNA polymerase (pink) begins transcribing leftward at PL and stops at the terminator at the end of N. The N mRNA is the only product of this transcription (c) Transcription in the presence of N. N (purple) binds to the nut region of the transcript, and also to NusA (yellow), which, along with other proteins not shown, has bound to RNA polymerase. This complex of proteins alters the polymerase so it can read through the terminator and continue into the delayed early genes.

31. Figure 8.16 Protein complexes involved in N-directed antitermination. (a) Weak, non-processive complex. NusA binds to polymerase, and N binds to both NusA and box B of the nut site region of the transcript, creating a loop in the growing RNA. This complex is relatively weak and can cause antitermination only at terminators near the nut site (dashed arrow). These conditions exist only in vitro. (b) Strong, processive complex. NusA tethers N and box B to the polymerase, as in (a); in addition, S10 binds to polymerase, arid NusB binds to box A of the nut site region of the transcript. This provides an additional rink between the polymerase and the transcript, strengthening the complex. NusG also contributes to the strength of the complex. This complex is processive and can cause antitermination thousands of base pairs downstream in vivo (open arrow).

32. Figure 8.17

33. Figure 8.18

34. Figure 8.19

35. Figure 8.20 Map of the PR' region of the λ, genome. The PR‘promoter comprises the -10 and -35 boxes. The qut site overlaps the promoter and includes the Q binding site upstream of the -10 box, the pause signal downstream of the transcription start site, and the pause site at positions +16 and +17.

36. 5 proteins (N, NusA, NusB, NusG and S10) collaborate in antitermination at theλ immediate early terminators.

37. NusA and S10 bind to RNA polymerase • N and NusB bind to the boxB and boxA regions • N and NusB bind to NusA and S10 • NusA stimulates termination by interfering with the binding between upstream part of the RNA hairpin and the core polymerase • N helps NusA bind RNA, preventing hairpin formation

38. Establishing Lysogeny The delayed early genes help establish lysogeny in two ways: • Some of the delayed early gene products are needed for integration of the phage DNA into the host genome; • The products of the cII and cIII genes allow transcription of the cI gene and therefore production of the λrepressor.

39. The promoter used for establishment of losogeny is PRE, which lies to the right of PR and cro. Transcription from this promoter goes leftward through the cI gene. The delayed early genes cII and cIII also participate in this process: CII, by directly stimulating polymerase binding to PRE and PI; CIII, by slowing degradation of CII.

40. Figure 8.21 Establishing lysogeny. Delayed early transcription from PRgives cII mRNA that is translated to CII (purple). CII allows RNA polymerase (blue and red) to bind to PRE and transcribe the CI gene, yielding repressor (green).

41. Figure 8.22 Binding of CII at the -35 box of both PRE and PI promoters of λ, phage. Ptashne and colleagues performed a DNase footprint analysis of the interaction between CII and two early λ. promoters, PRE (a) and PI (b), In (a), lanes 1-4 contained the following amounts of CII: lane 1, none; lane 2, 10 pmol; lane 3, 18 pmol; and lane 4, 90 pmol. In (b), lanes 1-4 contained the following amounts of CII: lane 1, none; lane 2, 18 pmol; lane 3, 45 pmol; lane 4,100 pmol. The CII footprint in both promoters includes the -35 box.

42. Figure 8.23

43. Summary Phage λ establishes lysogeny by causing production of enough repressor to bind to the early operators and prevent further early RNA synthesis. The promoter used for establishment of lysogeny is PRE, which lies to the right of PR and cro. Transcription from this promoter goes leftward through the cI gene. The products of the delayed early genes cII and cIII also participate in this process: CII, by directly stimulating polymerase binding to PRE; CIII, by slowing degradation of CII.

44. Autoregulation of cI Gene During Lysogeny • Repressor turns off interrupting lytic circle • PRM activating repressor synthesis • OR controls leftward transcription of cI • OR1+OR2 repressor

45. Figure 8.24 Maintaining lysogeny. (bottom) Repressor (green, made originally via transcription from PRE) forms dimers and binds cooperatively to OR1 and 2. The protein-protein contact between repressor on OR2 and RNA polymerase (red and blue) allows polymerase to bind to PRM and transcribe cI. (top) Transcription (from PRM) and translation of the cI mRNA yields a continuous supply of repressor, which binds to OR and OL and prevents transcription of any genes aside from cI.

46. Figure 8.25 Map of the DNA fragment used to assay transcription from cI and cro promoters. The numbers denote the distances (in bp) between restriction sites. The red arrows denote the in vitrocI and cro transcripts.

47. Figure 8.26 Analysis of the effect of λ repressor on cl and cro transcription in vitro. Ptashne and colleagues performed run-off transcription (which actually produced "stutter" transcripts) using the DNA template depicted in, Figure 8.25. They included increasing concentrations of repressor as shown at bottom. Electrophoresis separated the cl and cro stutter transcripts, which are identified at right. The repressor clearly inhibited cro transcription, but it greatly stimulated cl transcription at low concentration, then inhibited cl transcription at high concentration.

48. Figure 8.27

49. Figure 8.28 Principle of intergenic suppression to detect interaction between λ repressor and RNA polymerase. (a) With wild-type repressor and polymerase, the two proteins interact closely, which stimulates polymerase binding and transcription from PRM. (b) The repressor gene has been mutated, yielding repressor with an altered amino acid (red). This prevents binding to polymerase. (c) The gene for one polymerase subunit has been mutated, yielding polymerase with an altered amino acid (represented by the square cavity) that restores binding to the mutant repressor. Since polymerase and repressor can now interact, transcription from PRM is restored.

50. Summary