Chapter 8 Major Shifts in Prokaryotic Transcription

1. Chapter 8Major Shifts inProkaryotic Transcription

2. 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 associated with the host core RNA polymerase and change its specificity from early to middle to late. Modification of the Host RNA Polymerase

3. gp28: (1) diverts the host’s polymerase from transcribing host (2) switches from early to middle phage transcription gene gp33 and gp34: The switch from middle to late transcription occurs in much the same way, except that two polypeptides team up to bind to the polymerase core and change its specificity. RNA polymerase changes specificity

4. Fig. 8.1

5. Genetic evidence: Mutants of gp28, gp34 or 33 prevent early-to-middle, middle-to-late switch Biochemical data: Pero measured polymerase specificity by transcribing SP01 DNA in vitro with core (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 the following competitors, early SP01 RNA (green); middle RNA (blue); and late RNA (red). Look for the competition for the products:

6. B. subtilis can exist indefinitely in thevegetative, as long as conditions are appropriate for growth. Under starvation conditions, this organism forms endospores, that can survive for years until favorable conditions return Sporulation is a fundamental change Control of Transcription During Sporulation

7. 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 newσ factors that displace the vegetativeσ factor from the core RNA polymerase. Control of Transcription During Sporulation

9. At least three sigma 29 (sigma E), sigma 30 (sigma H), and sigma 32 (sigma C) in addition to sigma 43 (sigma A) are involved. More than one new sigma factors are involved in sporulation

10. The DNA region contains two promoters: a vegetative and a sporulation

11. In vitro transcription: Plasmid p213 + labeled nt+ Sigma E or sigma A, then hybridized the labeled RNA to southern blot containing EcoRI-HincII fragments of the plasmid Sigma E has some ability to recognize vegetative promoters

12. spoIID: well-characterize Sporulation gene. Rong prepared a restriction fragment containing the spoIID promoter and transcribed it in vitro with B. subtillis core RNA polymerase plus sigma E ( middle lane) or sigma B plus sigma C. Only the enzyme containing sigma E made the proper transcript.

13. Some prokaryotic genes must be transcribed under conditions where two differentσ factors are active. These genes contain two different promoters. This ensures their expression no matter which factor is present and allows for different control under different conditions. Genes with Multiple Promoters

14. Spo VG: transcribed by EB and E E. The last purification step was DNA-cellulose column chromatography. The polymerase activity in each fraction (red). The insert shows the results of a run-off transcription assay using a DNA with two SpoVG promoters.

15. Fig. 8.7

16. Purified sigma factors B and E by gel electrophoresis and tested them with core polymerase by the same run-off transcription assay.

17. Fig. 8.8

18. Fig. 8.9

19. When cells experience an increase in temperature, or a variety of other environmental insults, they mount a defense called the heat shock response. Molecular chaperones, proteases are produced. At least 17 new heat shock transcripts begins when at higher temperature (42 oC). This shift of transcription required -32 (H). The E. coli Heat Shock Genes

21. Phage  can replicate in either of two ways: lytic and lysogenic. Infection of E. coli by Phage 

22. A bacterium harboring the integrated phage DNA is called a lysogen The integrated DNA is called a prophage

24. Cro gene product blocks the transcription of  repressor CI N: antiterminator Extension of transcripts controlled by the same promoters. Q: antiterminator

25. The immediate early/delayed early/late transcriptional switching in the lytic cycle of phage is controlled by antiterminators. Lytic reproduction of Phage 

26. N utilization site NusA N: function by restricting the pause time at the terminator

27. Five proteins (N, NusA, NusB, NusG and S10) collaborate in antitermination at the immediate early terminators. Antitermination in the late region requires Q, which binds to the Q-binding region of the qut site as RNA polymerase is stalled just downstream of the late promoter. Antitermination

28. Highly conserved among Nut sites Help to stabilize the antitermination complex contains an inverted repeat

29. Gel mobility shift assay: binding between N and RNA fragment containing box B NusA+ N bound to the complex: Fig. 8.16 NusA, NusB, NusG, ribosomal S10 proteins interfere with antitermination

30. Highly conserved among Nut sites Help to stabilize the antitermination complex contains an inverted repeat

31. Nus A and S10 bind to RNA polymerase, and N and Nus B bind to the box B and box A regions of the nut site in the growing transcript.

32. Fig. 8.15

33. Fig. 8.17 Qut: Q utilization site Q binds directly to qut site not to the transcript

34. Phage  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. Establishing Lysogeny

35. Fig. 8.18 Delayed early transcription from PR gives cII mRNA that is transcribed to CII (purple), which allows RNA polymerase (blue and red) to bind to PRE and transcribe the cI gene

36. The promoter that is used to maintain lysogeny is PRM. It comes into play after transcription from PRE makes possible that burst of repressor synthesis that establishes lysogeny. This repressor binds to OR1 and OR2 cooperatively, but leave OR3 open. RNA polymerase binds to PRM,, in a way that contacts the repressor bound to OR2. Autoregulation of cI Gene During Lysogeny

37. Fig. 8.19

39. Run-off transcription (this construct does not contain OL, therefore, need to use very high concentration of repressor)

40. High levels of repressor can repress transcription from PRM, may involve interaction of repressor dimers bound to OR1, OR2 and OR3, with repressor dimers bound to OL1, OL2 and OL3 via DNA looping.

41. Intergenic suppressor mutation studies show that the crucial interaction between repressor and RNA polymerase involves region 4 of the σ subunit of the polymerase. RNA polymerase-repressor Interaction

42. Fig. 8.23

43. Fig. 8.24

44. Fig. 8.25

45. Depends on the outcome of a race between the products of the cI and cro genes. The winner of the race is further determined by the CII concentration, which is determined by the cellular protease concentration, which is in turn determined by environmental factors such as the richness of the medium. Determining the fate of a  Infection: lysis or lysogeny

46. Fig. 8.26

47. When a lysogen suffers DNA damage, it induces the SOS response. The initial event in this response is the appearance of a coprotease activity in the RecA protein. This causes the repressors to cut themselves in half, removing them from the  operators and inducing the lytic cycle. In this way, progeny  phages can escape the potentially lethal damage that is occurring in their host. Lysogen Induction

48. Fig. 8.27

49. Chapter 9DNA – Protein Interactions in Prokaryotes

50. Helix 2 of the motif (red) lies in the major groove of its DNA target