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Review of simple regulation Introduction to combinatorial control A prokaryotic example

Combinatorial control of a bacterial metabolic promoter. Review of simple regulation Introduction to combinatorial control A prokaryotic example The complex promoter acs P2 and its activation by CRP Introduction to nucleoid proteins (e.g. Fis and IHF)

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Review of simple regulation Introduction to combinatorial control A prokaryotic example

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  1. Combinatorial control of a bacterial metabolic promoter Review of simple regulation Introduction to combinatorial control A prokaryotic example The complex promoter acsP2 and its activation by CRP Introduction to nucleoid proteins (e.g. Fis and IHF) Fis anti-activates CRP-dependent activation How does IHF inhibit CRP-dependent activation? The experimental approach The mechanism (results and working model) Unanswered questions

  2. RNAP RNAP SS S70  &’  &’ • Bacterial cells use diverse mechanisms to regulate transcription • Sigma factors partition limited RNA polymerase (RNAP) • to different promoter subsets

  3. e.g., Activation by protein-protein contact  &’  &’ e.g., Repression by steric hindrance Bacterial cells use diverse mechanisms to regulate transcription 2. Transcription factors alter binding of RNAP to promoter

  4.  ’ NT  CT TTGACA TATAAT CCGACT TACACT Weak promoters Regulated by activation Weak promoter 1. fewer elements 2. less consensus UP -35 -10

  5. 4 2 1 1 3 3 5 5 TTGACA TATAAT 1) TF/DNA CCGACT TACACT 2) CRP/CTD 3) DNA/CTD 4) /CTD 5) /DNA (promoter) Recruitment by TF that increases affinity   ’ NTD  CTD UP -35 -10 TF Interactions that enhance transcription initiation

  6. 4 2 1 1 3 3 5 5 TTGACA TATAAT 1) TF/DNA 2) CRP/CTD 3) DNA/CTD 4) /CTD 5) /DNA (promoter) Repression by too much affinity   ’ NTD  CTD UP -35 -10 TF Interactions that enhance transcription initiation RNAP can’t break contacts used for TCC & TOC formation

  7. TF2 TF1 -35 -10 Combinatorial Control A simple example TF1 TF1 -35 -10

  8. Combinatorial Control a textbook, eukaryotic example

  9. Combinatorial Control a prokaryotic example

  10. Pi CoASH ADP ATP Acetate Switch Glucose 2 NAD 2 NADH + 2 H+ 2 ADP 2 ATP Pyruvate CoASH + NAD CO2 + NADH + H+ Acetyl-CoA AMP ACS CoASH Acetyl~P Acetyl-AMP PPi ACS ATP Acetate ACS = acetyl-CoA synthetase

  11. NADH TCA MDH NAD+ GAPDH glc NAD+ NADH NAD+ NADH CobB Ac ~ Acs PAT Inactive Regulation of Acs activity Acetyl-CoA AMP Acs CoASH Ac~AMP PPi Acs ATP Acetate

  12. glc [glc] ac~CoA Pta/Ack ace acs TRXN [ace] Regulation of acs transcription TCA ac~CoA Acs GROWTH ace TIME

  13. P1 pnrfA P2 nrfA nrfB acs actP nrfC yjcH -180 -226 -153 -267 -98 -59 IHF II IHF I FIS I IHF III FIS II FIS III P2 +1 CRP II CRP I P1 -122.5 -69.5 Pnrf

  14. 5 turns Beatty et al., 2003 acs transcription CRP (cAMP Receptor Protein)-dependent proximal P2 is the major promoter acs S70  &’ +1 CRP I -69.5 CRP II -122.5 P2 Higher affinity CRP I absolutely required for any activation Lower affinity CRP II doubles activity – with a twist!

  15. Bacterial cells use diverse mechanisms to regulate transcription 3. Regulation by histone-like proteins that organize bacterial chromatin

  16. Bacterial chromosome The traditional view “A tangled knot of DNA” Dr. Gopal Murti Science Photo Library Photo Researchers

  17. (transcriptionally inactive) Late stationary phase Lag phase trxn inactive Early stationary phase (transcriptionally active) Exponential growth trxn active Adapted from Kim et al., 2004 Bacterial chromosome The modern view Kim et al., 2004

  18. SP EP Ishihama, 1999 12 histone-like (nucleoid) proteins in E. coli Relative amounts change with phase of growth Fis (Factor for Inversion Stimulation) predominates during EP undetectable by ESP IHF (Integration Host Factor) present during EP Increases 4X 2nd most predominate in ESP

  19. acs S70  &’ +1 CRP I -69.5 CRP II -122.5 5 turns Nucleoid proteins influence acs transcription P2 • Fis • IHF • Browning et al., 2004

  20. A/T-rich facilitates bending GTGTAACAAATAACC Fis logo degenerate GCCGTTTATTTGCAC GCCCAAATACTAAAC 0 -7 +7 3 putative DNA sites forFis5’ of acs ORF Factor for Inversion Stimulation (Fis) homodimer bends DNA 50-90o Hengen et. al., 1997

  21. +1 acsP2 CRP II CRP I Fis II Fis III Fis sites overlap CRP sites Do they compete?

  22. - - - - - [CRP] - - - - - [Fis] GA 1 2 3 4 5 6 7 8 9 10 11 12 FIS I -270 The consequence? Fis inhibits CRP-dependent transcription (in vitro & in vivo) -213 CRP II CRP II -122 CRP II -106 FIS II -91 FIS II CRP I CRP I -72 CRP I -67 +1 FIS III -52 FIS III -98 -59 acsP2 Anti-activation -40 Fis II Fis III Fis and CRP interactions DNase I footprint analysis Fis out-competes CRP Fis + CRP resembles Fis alone

  23. glc TCA [glc] ac~CoA ac~CoA Pta/Ack Acs ace ace acs TRXN [ace] Regulation of acs transcription GROWTH TIME

  24. +1 -98 -59 acsP2 Fis II Fis III Anti-activation Fis sterically hinders CRP

  25. IHF III FIS I FIS II FIS III IHF I IHF II +1 P1 P2 CRP I CRP II Integration Host Factor (IHF) W W W W W W n n n n n n n n W A T C A A n n n n T T R T A T T T Ta c a t g c a cT t a C A At t g aT T AI AgT A T c t t c c t c t tT t TC A Ac a g c a T GII c A T A A c t g c a t g t t c c T C A A a g a aT T AIII

  26. IHF P1 IHF I IHF II IHF III [Fis] +1 - - - - - - - - - - [CRP] - - - - - - - - - - [IHF] - - - - - -226 -180 -153 P2 pnrfA Fis I IHF & Fis -238 IHF I IHF I IHF II IHF III Fis II Fis III Fis I -195 IHF II -167 IHF III -265 -226 -180 -153 -98 -59 -139 CRP II -122 IHF & CRP -106 Fis II CRP I CRP II -91 IHF I IHF II IHF III -72 CRP I -226 -180 -153 -122.5 -69.5 Fis III -52 IHF binds simultaneously with FIS and CRP DNase I footprint analysis

  27. CRP I CRP II IHF I IHF II IHF III Both FIS and IHF inhibit CRP-dependent activation 2 distinct mechanisms

  28. Approach

  29. CRP CRP How do you monitor promoter activity?“reporter” assay lac Z P2 CRP I

  30. How do you monitor promoter activity?“reporter” assay Activity (Miller Units) OD590

  31. Full length 444 236 205 155 P2 Acs Regulatory Region Nested Deletions FIS III FIS II FIS I CRP I IHF II CRP II IHF I IHF III WT / 2 WT WT / 6 WT

  32. CRP CRP 236 205 155 CRP CRP IHF P2 Acs Regulatory Region Mutations CRP I CRP II IHF III WT / 2 WT Activity WT/6 WT Activity (CRP and IHF dependent)

  33. CRP CRP CRP CRP IHF P2 Inhibitory Acs Regulatory Region Summary Activating Enhancing IHF III CRP I CRP II Activation requires CRP I, enhanced by CRP II Inhibition of activation requires IHF III & CRP II

  34. Mechanism

  35. CRP CRP σ Proteins have surfaces Rhodius RNAP AR 1 AR 1 Class I

  36. AR 2 CRP CRP AR 1 σ AR 3 Proteins have surfaces Rhodius RNAP Class II

  37. CRP CRP CRP CRP IHF P2 Inhibitory Acs Regulatory Region Summary Activating Enhancing IHF III CRP I CRP II We can use our knowledge concerning the surfaces of CRP and their interactions to determine mechanism

  38. CRP CRP CRPv pCRP AmpR The Experimental DesignComplementation CRP lac Z P2 CRP I

  39. 9800 1+ 2+ (WT) 8800 1+ 2- 7800 1+ 2- 3+ 6800 1- 2+ 3+ 5800 1- 2- 3+ 4800 1+ 2+ 3+ 3800 2800 1800 800 -200 0.01 0.1 1 10 Monitoring promoter activity of pacs155“reporter” assay Activity (Miller Units) OD590

  40. Monitoring promoter activity of pacs155a simplified version Activity (Miller Units)

  41. CRP CRP σ pacs155 is AR1-dependent classic Class I behavior RNAP AR 1 AR 1

  42. AR2 CRP CRP AR3 σ How can AR2 & AR3 inhibit? AR 2 RNAP AR 1 AR 1 AR 3 Later

  43. AR 1 CRP CRP AR 2 Activation requires AR1 Like pacs155 AR 3 CRP CRP P2 Inhibition requires both AR2 and AR3 Like pacs155 Key pacs205 CRP I CRP II

  44. AR 1 CRP CRP AR 2 Activation requires AR1 Like pacs155 AR 3 CRP CRP IHF P2 Inhibition requires either AR2 or AR3 Together = severe inhibition Key pacs236 CRP I CRP II IHF III

  45. AR 1 AR2 or AR3 enhances activation Opposite of pacs236 CRP CRP AR 2 AR 3 Activation requires AR1 Like pacs155 IHF P2 Key pacs236c2m CRP I CRP II IHF III Together AR2 and AR3 inhibit severely Like pacs236

  46. CRP CRP WT CRP has ONLY AR1 and AR2 IHF P2 Mechanism – a working model CRP I CRP II IHF III CRP I is high affinity, CRP II is low affinity Low concentration of CRP Fill ONLY CRP I = AR1-dependent, AR2-enhanced activation

  47. CRP CRP CRP CRP IHF P2 Mechanism – a working model CRP I is high affinity, CRP II is low affinity CRP I CRP II IHF III High concentration of CRP Fill BOTH CRP I & CRP II = AR2-dependent inhibition of AR1-dependent activation

  48. Fis and IHF bind & function independently FIS CRP CRP and IHF bind Simultaneously Low [ ] activates acs High [ ] inhibits TRXN Regulation of acs transcription Depends on [Fis] and [CRP] IHF GROWTH TIME

  49. The role of IHF To make the sense of regulation (+ or -) dependent on [CRP] Depends on AR2?!

  50. CRP CRP σ How can AR2 enhance or inhibit? AR 2 RNAP AR 1 AR 1 pacs205/236 have two dimers = Four AR2 However, pacs155 exhibits fundamental behavior Thus, likely due to one of two AR2 on proximal dimer

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