Chapter 16 Gene regulation in Prokaryotes

1. Chapter 16 Gene regulation in Prokaryotes

2. Gene expression is controlled by regulatory proteins • Regulatory proteins: positive regulators, or activators; and negative regulators, or repressors. • They are typically DNA-binding proteins that recognize specific sites at or near the gene they control.

3. Many promoters are regulated by activators that help RNA polymerase bind DNA and by repressors that block that binding • At many promoters, when RNA polymerase does bind to the promoter, it spontaneously undergoes a transition to the open complex and initiates transcription, called basal level. • The site on DNA where a repressor binds is called an operator. • Some activators help polymerase bind the promoters. This mechanism, called recruitment, is an examples of cooperative binding of protein to DNA.

4. Some activators work by allostery and regulate steps after RNA polymerase binding • Some promoters require activators to stimulate the transition from closed to open complex. • Activators that stimulate this kind of promoter work by triggering a conformation change in either RNA polymerase or DNA. • This mechanism is an example of allostery. • One activator, NtrC, interacts with the RNA polymerase bound in a closed complex at the promoter and stimulates transition to the open complex. It is an example of σ54 holoenzyme transcription.

5. Allosteric activation of RNA polymerase

6. Action at a distance and DNA looping • NtrC activates a promoter “from a distance”: its binding sites are normally located about 150 bp upstream of the promoter. • One way to help bring distant DNA sites closer together (and so help looping) is the binding of other proteins to sequences between those sites.

7. DNA-bending protein • There are cases in which a protein binds between an activator binding site and the promoter and helps the activator interact with polymerase by bending the DNA

8. Cooperative binding and allostery have many roles in gene regulation • Simple cooperative binding: the activator interacts simultaneously with DNA and with polymerase and so recruits the enzyme to the promoter. • Allostery is not only a mechanism of gene activation, it is also often the way regulators are controlled by their specific signals. • A typical bacterial regulator can adopt two conformations- in one it can bind DNA, in the other cannot, depends on the presence of a signal molecule.

9. Regulation of transcription initiation:examples from bacteria • The lac Operon: It contains three structural genes – genes that code for proteins : -galactosidase (lacZ), galactoside permease (lacY), and galactoside transacetylase (lacA). • They all are transcribed together on one m RNA, called a polycistronic message, starting from a single promoter.

10. The mechanism of Repression • The lac operator overlaps the promoter, and so repressor bound to the operator physically prevents RNA polymerase from binding to the promoter. • Negative Control of the lac Operon • Repressor-operator Interactions

11. Positive Control of the lac Operon • It is mediated by a factor called catabolite activator protein (CAP) in conjunction with cyclic AMP, to stimulate transcription. • Sensed the lack of glucose, increase of cAMP.

12. CAP is dimeric and binds to 22 bp operator sequences, accelerates the initiation of transcription at these promoters.

13. CAP has separate activating and DNA binding surfaces

14. CTD

15. CAP and lac repressor bind DNA using a common structural motif • Recognition of specific DNA sequences is achieved using a conserved region of a helix-turn-helix. • This domain is composed of two alpha helices, one is the recognition helix.

17. Lac repressor binds as a tetramer to two operators: in such case, the interventing DNA loops out to accommodate the reaction.

18. The activities of Lac repressor and CAP are controlled allosterically by their signals • It is allolactose (rather than lactose itself) that controls Lac repressor. • Allolactose binds to Lac repressor and triggers a change in the shape (conformation) of the protein. • Glucose lowers the intracellular concentration of a small molecule, cAMP. This molecule is the allosteric effector for CAP. • Only when CAP is complexed with cAMP does the protein adopt a conformation that binds DNA.

19. Partial diploid cells show that functional repressors work in trans.

20. Partial diploid cells show that operators work only in cis.

21. Alternative  factors direct RNA polymerase to alternative sets of Promoters • Heat shock  factor is 32. When E. coli is subject to heat shock, the amount of this new  factor increases in the cell, and displaces  70 from a proportion of RNA polymerases. •  54 in the cells is required to transcribe genes involved in nitrogen metabolism.

22. 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.

23. NtrC and MerR: transcriptional activators that work by allostery rather than by recruitment • The majority of activators work by recruitment. • Two exceptions: NtrC and MerR. • In the case of activators that work by allosteric mechanisms, polymerase initially binds the promoter in an inactive complex. The activator triggers an allosteric change in that complex. • NtrC induces a conformational change in the enzyme that triggers open complex formation.

24. NtrC has ATPase activity and works from DNA sites far from the gene • NtrC binds to each site as a dimer, and through protein-protein interactions between the dimers, binds to the four sites in a highly cooperative manner

25. MerR activates transcription by twisting promoter DNA • MerR binds to a sequence located between the -10 and -35 regions of the merT promoter. • MerR binds to the opposite face of the DNA helix from that bound by RNA polymerase. • When MerR binds to Hg2+, the protein undergoes a conformational change that causes the DNA in the center of the promoter to twist. • It is an example of altering the conformation of DNA in the vicinity of the prebound enzyme.

27. AraC and control of the araBAD operon by antiactivation • The promoter of the araBAD operon from E. coli is activated in the presence of arabinose and the absence of glucose and directs expression of genes encoding enzymes required for arabinose metabolism. • Activator AraC adopts different conformations in the presence or absence of arabinose.

28. Examples of gene regulation at steps after transcription initiation • In E. coli the five contiguous trp genes encode enzymes that synthesize the amino acid tryptophan. • Tryptophan acts as a corepressor, not an inducer. • When tryptophan is present, it binds to Trp repressor and induces a conformational change in that protein and enables it to bind the trp operator.

29. Amino acid biosynthetic operons are controlled by premature transcription termination • Once polymerase has initiated a trp mRNA molecule, it does not always complete the full transcript. • The mechanism overcomes the premature transcription termination is called attenuation.

32. The case of phage : layers of regulation • Phage  can replicate in either of two ways: lytic and lysogenic. • A bacterium harboring the integrated phage DNA is called a lysogen • The integrated DNA is called a prophage. • The switch from lysogenic to lytic growth is called lysogenic induction.

33. Alternative patterns of gene expression control lytic and lysogenic growth •  has a 50-kb genome and some 50 genes. • Promoters in the right and left regions of phage 

34. Promoters in the right and left control regions of phage  PR and PL ( stand for rightward and leftward promoter) are strong promoters. PRM ( promoter for repressor maintenance), transcribing only the cI gene, is a weak promoter and only directs efficient transcription when an activator is bound just upstream.

35. Transcription in the  control regions in lytic and lysogenic growth cI gene encodes  repressor

36. Regulatory proteins and their binding sites • The cI gene encodes  repressor, a protein of two domains joined by a flexible linker region. • As an activator,  repressor works like CAP, by recruitment.  repressor’s activating region is in the N-terminal domain of the protein. • Cro (control of repressor and other things) only represses transcription.

37. and cro can each bind to any one of six operators. OR1,OR2 and OR3 in the right operators are similar in sequences but not identical. The affinities of these various interactions, however, are not all the same.

38. repressor binds to operator sites cooperatively • repressor at OR1 helps repressor bind to the lower affinity site OR2 by cooperative binding.

39. Repressor and Cro bind in different patterns to control lytic and lysogenic growth

40. During lysogeny, PRM is on , while PR and PL are off. 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. For lytic growth, a single Cro dimer is bound to OR3; this is overlaps PRM and so Cro represses that promoter.

42. Lysogenic induction requires proteolytic cleavage of  repressor • When a lysogen suffers DNA damage, it induces the SOS response. • The initial event in SOS response is the appearance of a coprotease activity in the RecA protein and then it stimulates autocleavage of LexA, that represses genes encoding DNA repair enzymes. • This causes the repressors to cut themselves in half, removing them from the  operators and inducing the lytic cycle.

43. Another activator, λcII, controls the decision between lytic and lysogenic growth upon infection of a new host

44. The promoter used for establishment of lysogeny is PRE. Delayed early transcription from PR gives cII mRNA that is transcribed to CII, which allows RNA polymerase to bind to PRE and transcribe the cI gene PRM comes into play after transcription from PRE makes possible that burst of repressor synthesis that establishes lysogeny.

45. Growth conditions of E. coli control the stability of CII protein and thus the lytic/lysogenic choice • When the phage infects healthy bacterial cells, it tends to propagate lytically.

46. CII is a very unstable protein and is degraded by a specific protease called FtsH. If growth is good, FstH is very active, then CII is low. In poor growth conditions, slow degradation of CII leads to lysogeny.

47. N utilization Transcription antitermination in λ development • N and Q only work on genes that carry particular sequences. • Five proteins (N, NusA, NusB, NusG and NusE) collaborate bind to RNA transcribed from DNA containing a nut (for N utilization) sequence in antitermination in the early operons of . • Antitermination in the late region requires Q, which binds to DNA sequences (QBE) between the –10 and –35 regions of the late gene promoter (PR’).

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