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Welcome to Introduction to Bioinformatics Friday, 19 September 2014

Welcome to Introduction to Bioinformatics Friday, 19 September 2014. Scenario 2: Simulation Finding biologically important sites in DNA How to avoid being fooled by imposters?. Scenario. Gene regulation. Scenario 2. Finding biologically important sites in DNA.

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Welcome to Introduction to Bioinformatics Friday, 19 September 2014

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  1. Welcome toIntroduction to BioinformaticsFriday, 19 September 2014 Scenario 2: Simulation Finding biologically important sites in DNA How to avoid being fooled by imposters? • Scenario • Gene regulation

  2. Scenario 2 Finding biologically important sites in DNA

  3. You: A typical grad student

  4. You: A typical grad student

  5. Your object of study: Cyanobacteria

  6. Critical position in food web CO2 sugarN2 ammoniaH2O electrons Your object of study: Cyanobacteria How do they do it?

  7. heterocysts sucrose N2 fixation in cyanobacteria N2 CO2 O2 Matveyev and Elhai (unpublished)

  8. heterocysts sucrose NH3 N2 fixation in cyanobacteria NH3 N2 O2 CO2 Matveyev and Elhai (unpublished)

  9. Differentiation in cyanobacteria -NH3 ? ? ? ? ? Heterocysts

  10. Response to environment How do bacteria respond to the environment? From gene to protein DNA RNA protein

  11. How do bacteria respond to the environment? From gene to protein RNAPol DNA P RNA protein

  12. NH3 glutamine α-ketoglutarate How do cyanobacteria respond to NH3? From gene to protein High N Low N DNA binding protein, NtcA RNAPol DNA Binding site P No RNA

  13. NH3 glutamine α-ketoglutarate How do cyanobacteria respond to NH3? From gene to protein High N Low N DNA binding protein, NtcA RNAPol DNA Binding site P No RNA

  14. RNA protein How do cyanobacteria respond to NH3? From gene to protein Low N RNAPol NtcA DNA Binding site P α-ketoglutarate

  15. Differentiation in cyanobacteria -NH3 ? ? ? ? ? Heterocysts

  16. Differentiation in cyanobacteria -NH3 Activates NtcA (Nitrogen Control) ? ? ? Heterocysts

  17. Differentiation in cyanobacteriaWhat DNA site does NtcA bind to? RNAPol NtcA Binding site P

  18. Differentiation in cyanobacteriaWhat DNA site does NtcA bind to?

  19. mRNA GTA…(8)…TAC …(20-24)…TAnnnT Differentiation in cyanobacteriaWhat DNA site does NtcA bind to? RNAPol NtcA Binding site P Herrero et al (2001) J Bacteriol 183:411-425

  20. HetQ -N NtcA ??? Position in cell cycle Level of PatS Level of HetN Differentiation in cyanobacteriaIntegration of signals through HetR Genes needed for differentiation HetR Master regulator StrategyPCR out hetQRandom mutagenesisLook for effects on HetR expression/activity

  21. Differentiation in cyanobacteriaFind primers to PCR out hetQ cctatctccgccctatggcgatttgggcaatatatttgatgattggttag ...hypothetical ttgtcagttgtcagacgtagtagcgcgtctagtctaatgtgttgttatat protein tatttgctactagaaatgaggagagggttatttttctcactgcttcccaa ttctatgagaatataaaattttccttaagtttctcatggcaataatggaa aaaaccgaccattctgatgaataagtccggttttttccaaaaaatatttt tgctttttcgctttatttatctatatttccaagttttagtacatcggtga ggggtgacaactatcttgccaatattgtcgttattgttaggttgctatcg gaaaaaatctgtaacatgagatacacaatagcatttatatttgctttagt atctctctcttgggtgggattctgcctgcaatttaaaaaccagtgttaac aattttcggctttattttccgggagttaaatcaaccaagggaaaatgtaa ctaatgtttaaatatcttcggatacacacaaagtaaaaccaatttttaca gatgtcgatgttgctcacattttttagaaatattactaaattaaaaatgt tattaaatttatgttcatagagaaccttttccaaataaaaaaataatttt cctgatgttttaagaaaattactgttgttataaattaaaggtgattcaac aaaatatagatagttctttcaataactatctacttttaccattaagtgaa cttactcatgaataatcaacaggaattaaaaataaagttcatgaatactg gttaaagattcagtaaagtttgaggaaataccggaataaatttccaccca aatatgattttttaaaagatacattggcagtacattaaaatgccgatgtt agataaatttgccttcatagctgttatctatttgctcagaactaagccaa gagtttacacaccaaacagaaattaaactatgaatccctcttcgtcgtta hetQ...

  22. Differentiation in cyanobacteriaFind primers to PCR out hetQ cctatctccgccctatggcgatttgggcaatatatttgatgattggttag ...hypothetical ttgtcagttgtcagacgtagtagcgcgtctagtctaatgtgttgttatatprotein tatttgctactagaaatgaggagagggttatttttctcactgcttcccaa ttctatgagaatataaaattttccttaagtttctcatggcaataatggaa aaaaccgaccattctgatgaataagtccggttttttccaaaaaatatttt tgctttttcgctttatttatctatatttccaagttttagtacatcggtga ggggtgacaactatcttgccaatattgtcgttattgttaggttgctatcg gaaaaaatctgtaacatgagatacacaatagcatttatatttgctttagt atctctctcttgggtgggattctgcctgcaatttaaaaaccagtgttaac aattttcggctttattttccgggagttaaatcaaccaagggaaaatgtaa ctaatgtttaaatatcttcggatacacacaaagtaaaaccaatttttaca gatgtcgatgttgctcacattttttagaaatattactaaattaaaaatgt tattaaatttatgttcatagagaaccttttccaaataaaaaaataatttt cctgatgttttaagaaaattactgttgttataaattaaaggtgattcaac aaaatatagatagttctttcaataactatctacttttaccattaagtgaa cttactcatgaataatcaacaggaattaaaaataaagttcatgaatactg gttaaagattcagtaaagtttgaggaaataccggaataaatttccaccca aatatgattttttaaaagatacattggcagtacattaaaatgccgatgtt agataaatttgccttcatagctgttatctatttgctcagaactaagccaa gagtttacacaccaaacagaaattaaactatgaatccctcttcgtcgtta hetQ...

  23. Differentiation in cyanobacteriaFind primers to PCR out hetC ttgtcagttgtcagacgtagtagcgcgtctagtctaatgtgttgttatat tatttgctactagaaatgaggagagggttatttttctcactgcttcccaa ttctatgagaatataaaattttccttaagtttctcatggcaataatggaa aaaaccgaccattctgatgaataagtccggttttttccaaaaaatatttt tgctttttcgctttatttatctatatttccaagttttagtacatcggtga ggggtgacaactatcttgccaatattgtcgttattgttaggttgctatcg gaaaaaatcTGTAacatgagaTACAcaatagcatttatatttgctttagt atctctctcttgggtgggattctgcctgcaatttaaaaaccagtgttaac aattttcggctttattttccgggagttaaatcaaccaagggaaaatgtaa ctaatgtttaaatatcttcggatacacacaaagtaaaaccaatttttaca gatgtcgatgttgctcacattttttagaaatattactaaattaaaaatgt tattaaatttatgttcatagagaaccttttccaaataaaaaaataatttt cctgatgttttaagaaaattactgttgttataaattaaaggtgattcaac aaaatatagatagttctttcaataactatctacttttaccattaagtgaa cttactcatgaataatcaacaggaattaaaaataaagttcatgaatactg gttaaagattcagtaaagtttgaggaaataccggaataaatttccaccca aatatgattttttaaaagatacattggcagtacattaaaatgccgatgtt agataaatttgccttcatagctgttatctatttgctcagaactaagccaa gagtttacacaccaaacagaaattaaactatgaatccctcttcgtcgtta hetC... GTA…(8)…TAC

  24. Differentiation in cyanobacteria ttctatgagaatataaaattttccttaagtttct aaaaccgaccattctgatgaataagtccggtttt tgctttttcgctttatttatctatatttccaagt ggggtgacaactatcttgccaatattgtcgttat gaaaaaatctGTAacatgagaTACacaatagcatttatatttgcttTAgtaTctctctcttgggtggg …(20-24)…TAnnnT GTA…(8)…TACNtcA binding site Promoter

  25. Differentiation in cyanobacteriaIntegration of signals through HetR ??? HetQ -N ??? NtcA ??? Genes needed for differentiation Position in cell cycle HetR Level of PatS Level of HetN Master regulator Stockholm

  26. How to proceed? • Choice #1 • Publish • Grant proposals • Build a career • Likely result • Reviewers trash MS: too speculative

  27. How to proceed? • Choice #2 • Forget about it • Back to PCR • Likely result • Sometimes miss spectacular finding

  28. How to proceed? • Choice #3 • Forget about PCR • Do backbreaking NtcA binding studies I'd knock out NtcA, reintroduce it in plasmid to nostoc, and do RT-PCR to check gene expression. • Likely result • Might demonstrate binding of NtcA • Risky, may lose many months

  29. How to proceed? • Choice #4 • Determine whether site is likely to be real How? N! . . .a! (N-a)! • High school math approach

  30. How to proceed? • Choice #4 • Determine whether site is likely to be real How? BIOINFORMATICS • Simulation • Exhaustive pattern search

  31. Regulatory Protein and their Binding Sites What do we talk about? • Significance of palindromes (SQ7 and topic H) • Nature of regulation (through gene fusions (SQ8) • Gene fusions: e.g. ntcA / lacZ(SQ8) • How many promoters? CRP-binding sites? (SQ5) • Simulations? • Why do them? (SQ10) • Pitfalls? (SQ9)

  32. Backwards = forwards GCTATCG • DNA is double stranded ROTATOR TTAATGTGAGTTAGCTCACTCATTAATTACACTCAATCGAGTGAGTAA Regulatory Protein and their Binding Sites Palindromic sequences What is it? What about with DNA?

  33. Backwards = forwards GCTATCG ROTATOR Regulatory Protein and their Binding Sites Palindromic sequences What is it? What about with DNA? • DNA is double stranded • DNA is redundant TTAATGTGAGTTAGCTCACTCATTAATTACACTCAATCGAGTGAGTAA

  34. Backwards = forwards GCTATCG ROTATOR TTAATGTGAGTTAGCTCACTCATT AATGAGTGAGCTAACTCACATTAA Regulatory Protein and their Binding Sites Palindromic sequences What is it? What about with DNA? • DNA is double stranded • DNA is redundant • DNA has direction (read 5’->3’) 5’- -3’ 3’- -5’ TTAATGTGAGTTAGCTCACTCATTAATTACACTCAATCGAGTGAGTAA

  35. TAT GGCATGCTAGCTTAAT TCATTAATTA AGTAACGTACGATCGG TAT DNA: cruciform RNA: stem/loop Regulatory Protein and their Binding Sites Palindromic sequences 5’- -3’ 3’- -5’ TTAATGTGAGTTAGCTCACTCATTAATTACACTCAATCGAGTGAGTAA

  36. UAU GGCAUGCUAGCUUAAU UCAUU tRNA DNA: cruciform RNA: stem/loop Regulatory Protein and their Binding Sites Palindromic sequences 5’- -3’ 3’- -5’ TTAATGTGAGTTAGCTCACTCATTAATTACACTCAATCGAGTGAGTAA

  37. TTAATGTGAGTTAGCTCACTCATT NNNNNNNNNNNNNNNNNNNNNNNNNNNNNN NNNNNNNNNNNNNNNNNNNNNNNNNN AATGAGTGAGCTAACTCACATTAA recognizes GTGAGTT Regulatory Protein and their Binding Sites Palindromic sequences

  38. TTAATGTGAGTTAGCTCACTCATT NNNNNNNNNNNNNNNNNNNNNNNNNNNNNN NNNNNNNNNNNNNNNNNNNNNNNNNN AATGAGTGAGCTAACTCACATTAA Regulatory Protein and their Binding Sites Palindromic sequences

  39. Regulatory Protein and their Binding Sites Palindromic sequences TTAATGTGAGTTAGCTCACTCATT NNNNNNNNNNNNNNNNNNNNNNNNNNNNNN NNNNNNNNNNNNNNNNNNNNNNNNNN AATGAGTGAGCTAACTCACATTAA

  40. Regulatory Protein and their Binding Sites Palindromic sequences TTAATGTGAGTTAGCTCACTCATT NNNNNNNNNNNNNNNNNNNNNNNNNNNNNN NNNNNNNNNNNNNNNNNNNNNNNNNN AATGAGTGAGCTAACTCACATTAA

  41. Regulatory Protein and their Binding Sites Palindromic sequences TTAATGTGAGTTAGCTCACTCATT NNNNNNNNNNNNNNNNNNNNNNNNNNNNNN NNNNNNNNNNNNNNNNNNNNNNNNNN AATGAGTGAGCTAACTCACATTAA

  42. Regulatory Protein and their Binding Sites Palindromic sequences TTAATGTGAGTTAGCTCACTCATT NNNNNNNNNNNNNNNNNNNNNNNNNNNNNN NNNNNNNNNNNNNNNNNNNNNNNNNN AATGAGTGAGCTAACTCACATTAA recognizes GTGAGTT

  43. Regulatory Protein and their Binding Sites Palindromic sequences TTAATGTGAGTTAGCTCACTCATT NNNNNNNNNNNNNNNNNNNNNNNNNNNNNN NNNNNNNNNNNNNNNNNNNNNNNNNN AATGAGTGAGCTAACTCACATTAA

  44. Regulatory Protein and their Binding Sites Palindromic sequences TTAATGTGAGTTAGCTCACTCATT NNNNNNNNNNNNNNNNNNNNNNNNNNNNNN NNNNNNNNNNNNNNNNNNNNNNNNNN AATGAGTGAGCTAACTCACATTAA

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