Transcription complex assembly: 1. The basic mechanism. 2. How do activators fit in?

1. Transcription complex assembly: 1. The basic mechanism. 2. How do activators fit in?

2. Recall from before: beta, beta’ Constant pol footprint alpha sigma spacer up -35 -10 katG oxyR site m-RNA -100 -80 -60 -40 -20 +1 +20 +40 Repression zone activation zone

3. Recall also that many promoters do not require activators. What is their pathway of of complex assembly? Historically, divided into binding and isomerization phases. Evidence for isomerization includes: 1970s - As complexes assemble they become resistant to the inhibitor heparin. 1980s - Kinetic and footprinting studies reveal intermediate complexes. Chemical probing shows that the DNA opens over the start site. 1990s - Several different structures are solved. Some represent intermediates.

4. spacer -35 -10 The T7 A1 promoter has these interactions, which are sufficient for assembly without an activator. beta, beta’ Constant pol footprint alpha sigma r4 r2 up m-RNA -100 -80 -60 -40 -20 +1 +20 +40 Sclavi et al PNAS (2005) 102:4706 used rapid kinetic footprinting to follow how DNA is protected during the time course of complex assembly.

5. First binds up element and -35 Then extends towards +1 Finally binds downstream

6. DNA opens and goes into beta/beta’ jaws Then sigma r2 binds -10 Their model for open complex formation (includes other data) Heparin-resistant open complex Closed complexes Initial binding via alpha and sigma r4 on upstream elements isomerization

7. What approaches are used to find out how activators work? General idea : they compensate for lack of DNA elements. Genetics - which parts of activators and RNA polymerase? Biochemistry - same and which steps are affected? Informatics - what parts are conserved? Structure analysis - integrate above

8. Txn + OxyR up up in general, oxyR sites are adjacent to the place where the pol alpha subunits bind. They are sited at the very common -62 location. up up up down

9. Alpha (dimer) has N and C terminal domains connected by a linker CTD NTD beta beta’ CTD NTD Mol Microbiol (1993);7:859-64 Delete CTD and do transcription. Basal level OK but OxyR no longer activates. (example in a few minutes) Conclusion CTD is likely the target of oxyR

10. Isolation of CTD mutants that fail to support oxyR activation Map and sequence these mutants If activation fails then the lacZ-dependent color change will not occur - mutants are white Plasmid library expressing mutant forms of the alpha CTD katG-lacz fusion on the chromosome Mutant pols fail to activate the katG promoter if they fail to contact oxyR Wt turns cells red (lacZ indicator) Tao et. al (1995) J. Bact. 177. pp. 6740-44

11. Find 2 patches of point mutants near alpha 269 and 299. Modest 2-3x activation defects in vivo Now ask: Do they have activation defects in vitro?

12. CTD mutants fail to be activated properly in vitro. Basal transcription is not affected. wt mutants

13. OxyR:CTD interactions presumably stabilize RNAP on DNA. (promoter elements cannot do this alone at target promoters). beta oxyR 298 268 -35 -10 sigma beta' -60 -40 +1 activation zone Why is there an activation zone for oxyR and others? Its where such activators can reach the CTD.

14. Another activator- crp. The lac promoter requires crp because the basal elements are an imperfect match 4/6 5/6 These are the basal elements X 18 spacer Polymerase alpha subunits Sigma r4 Sigma r2 Crp activation site (glucose response) Lac repressor site (lactose response) And where crp and repressor binding sites conform to expectations

15. Signaling pathway where c-Amp activates crp only when external glucose is absent. (makes sense since glu is the preferred carbon source) A membrane complex containing coupled glucose transporter and adenylate cyclase glucose ATP X glucose 6-phosphate 3’ 5’ cyclic Amp (lactose) c-AMP concentrations drop as glucose is transported. c-AMP is needed to bind and change crp to its active conformation. Therefore, glucose shuts down lac and other operons by inactivating a required activator.

16. crpis a symmetric dimer So its binding site contains an inverted repeat with 2 identical half-sites Most bacterial activator and repressor sites have this symmetry because the multimers are symmetric. 2 DNA half-sites Crp dimer with 2 DNA “reading heads” C-AMP binds here and freezes this conformation

17. A brute force approach to find the alpha CTD determinants for crp activation. Savery et al (2002) J. Bact, Vol. 184 p. 2273-2280,, Change every residue to alanine and look for defective transcription on promoter where crp binds at -62.

18. Ctd aa# Several alpha CTD sites are found, including ones near 261, 271 and 288 Recall oxyR uses sites at 269and 299. So both use 269/271 + each has other site(s) to use on alpha.

19. Now turn to the donor site on crp (the one on oxyR is not known). How to find positive control mutants, ones that bind DNA but fail to activate? Next slide outlines the “pc” genetic screen. Zhou Y, Zhang X, Ebright RH Proc Natl Acad Sci U S A (1993)90:6081-5

20. Screen for activation domain mutants (positive control mutants) Strain x82 lacks crp and its redindicator is bleached white by lac expression. It contains 2 fusions as shown below. Crp is expressed from a plasmid and the crp gene is mutagenized. The screen finds dead mutants that still bind DNA. Crp Site (-62) Rbs basal elements Rbs genes Rbs expression bleaches red indicator to white 1. Lac UV5 elements Lac genes lac expression bleaches red indicator to white 2. Artificial crp site for repression type of crp fusion 1 fusion 2 color Is bleached white Crp+ bleaches white stays red Non-binding Crp- stays red bleaches white Is bleached white DNA-Binding Crp- stays red stays red Not bleached So stays red

21. Results: Mutants form an activation patch (AR1) on crp structure. This presumably touches one of the patches on alpha. (elaborate on next few slides). This shows the crp dimer bound to DNA. The patch is shown in only 1 subunit.

22. AT AT Subsequently Benoff et al Science (2002) 297:1562-6 proposed a structure of Crp + Alpha CTD + DNA. The next few slides show aspects of this structure. crp DNA used: binds crp These should bring in alpha CTD (up element sequences)

23. AT AT Structure shows alpha interacting with crp and with DNA The unit cell contained One crp dimer and 4 CTDs. See again on next slide

25. Just a reminder that this stabilizes pol An activation complex - The interface is small, but specific. 287 AR1 Dark blue is crp AR1. Yellow is CTD receptor 287/288 Red is CTD (265) that binds DNA up element (White 261may bind sigma in other contexts?)

26. All of this data comes from crp binding at a site near -60. It turns out that activation from a site near -40 is somewhat different. So the genetic and biochemical studies were also done using a promoter with a -40 site. A different surface was found and called "AR2" to distinguish it from AR1. The next experiment shows that a different receptor site is used from -40.

27. AR2 Interacts with the alpha N-Terminal Domain ss crp * pol X-link ss crp * reduce SH crp SH * Bifunctional x-linker transfers label from crp to alpha subunit of polymerase When alpha is proteolyzed it is the N-terminal domain that is labeled

28. AR2 was mapped by genetics. (some evidence that AR3 binds sigma - see sigma receptor later)

29. Some arrangements for which evidence exists FIG. 5. Model for class I and class II CRP-dependent promoter complexes. (A) At a class I CRP-dependent promoter with a CRP binding site centered at approximately -61.5 [e.g., CC(-61.5) or lacP1], the CTD 287 determinant interacts with AR1 of the downstream subunit of the CRP dimer, the 265 determinant interacts with DNA, and the 261 determinant interacts with (4). The relative contribution of the CTD 265 determinant to complex formation (hatched) varies, depending on promoter sequence. (B) At a class II CRP-dependent promoter [e.g., CC(-41.5) or galP1], the CTD 287 determinant interacts with AR1 of the upstream subunit of the CRP dimer bound at -41.5, and the 265 determinant interacts with DNA. A second activating region of CRP (AR2) interacts with NTD at class II CRP-dependent promoters (21).

30. What does the activator contact do for transcription? One question: After crp drives RNA polymerase into an open complex is it still needed during initiation? Many experiments use the inhibitor heparin, a polyanion that inactivates pol unless it is in an open complex at the promoter. See next 2 slides.

31. Simplified recognition pathway - use of heparin act pol Heparin-sensitive polymerase in a closed complexes Polymerase becomes heparin-resistant in open complex

32. The EMBO Journal Vol. 17,pp. 1759-1767, 1998 Result: Crp not needed for initiation after open complexes form DNA label in band shift Blot for crp transcription

33. Does activator contact simply recruit pol via binding? pol act "binding" (Kb) "isomerization"(K2 or Kf) recruitment

34. How to distinguish binding and isomerization effects? Use kinetics or footprinting. Footprinting: is the polymerase on the DNA prior to adding activator. If not, then activator must be influencing binding (it recruits). Many studies, including crp at the lac promoter, clearly show recruitment. Kinetics: measure binding and isomerization constants. Repeat with activator and infer the mechanism.

35. RNAP + DNA Closed complex Open complex Kb K2 Up 30x No change Crp at gal No change Up 17x Lambda C1 at Prm Selected summary of (old) kinetic data eg McClure W. PNAS (1997) 94:4691 binding isomerization Kb K2 Changed kinetics due to activator Most systems show an effect on binding, but some show an additional isomerization effect. The lambda activator shows only an isomerization effect.

36. Brief overview of the lambda c1 protein Activates when near -40. Represses when in the repression zone. Genetics suggest a sigma subunit contact. Extensive structural information - First look at docking of separately determined structures into an activation complex. Then look at an experimental complex.

37. Predicted docking of C1, DNA and pol structures Note predicted activator:sigma region4 contact that would stabilize RNA polymerase at the promoter. The next slide shows this region in an experimental (not docked) structure.

38. Molecular Cell, Vol 13, 45-53, 16 January 2004 -real structure Is there evidence for “isomerization effect? Claim on next 2 slides - convincing? The major interface is very small, but very specific. Lambda C1 Sigma r4

39. They compared the actual structure to a docked model derived from known structures of the individual components. Claim: all parts must undergo slight distortions to fit together: isomerization of complex. Claim of “re-modeled complex (B) Comparison of binary and ternary complexes. The cI dimer from the ternary complex is colored green, s4 orange. Relative movements of the cI and s4 monomers from the binary to ternary complexes are denoted by thethick arrows See also next slide

40. This shows induced distortion in the DNA. Near -40; can this be an isomerization effect? A productive complex (in which lcI and s4 make favorable protein-protein interactions) and a hypothetical nonproductive complex (in which lcI and s4 are misaligned, as is predicted to occur in the closed complex) are shown. RNAP holoenzyme is shown as a molecular surface (except s4 is shown as a backbone worm), color coded as follows: aI, aII, w, gray; b, cyan; b¢, pink; s, orange. The DNA is shown as phosphate backbone worms. For the productive complex, the DNA template strand is dark green, nontemplate strand light green, except the -35 element is yellow and the lcI operator is magenta. For reference, every fifth base pair between -15 and -60 (with respect to the transcription start site at +1) is shown schematically, and the positions are labeled in the scale above. The lcI dimer is shown as a green backbone worm. For the nonproductive complex, the DNA and lcI dimer are shown as blue backbone worms, a nd the DNA is shown only for the -35 element and upstream. The green and blue arrows indicate the upstream path of the DNA for the productive and nonproductive complexes, respectively.

41. You decide - does the structure support the kinetic data? pol act "binding" (Kb) "isomerization"(K2 or Kf) recruitment

42. Write a page or so on: Are the lambda c1:sigma structural studies consistent with the kinetic data? Could the effect be at “recruitment?” How would you test?

43. alpha ctd ntd beta Activators can touch multiple targets sigma beta' Activators must bind within the activation zone so they can reach the CTD. They must bind at certain positions so that the rotational orientation will allow proper contact.

44. In general, activated promoters typically have a poor enough match to consensus that polymerase won’t bind. Sometimes the bound RNA polymerase needs activator to change to its active form (details still lacking?) Therefore transcription is dependent on activator spacer Polymerase alpha subunits Sigma r4 Polymerase in active form Sigma r2 They bind here so they can touch polymerase And repressors bind here so they can block polymerase binding (anywhere the polymerase footprints) OPERATORS

45. Any of these may compensate for poor DNA elements. This is how activators work. activator -35 -10 alpha