Welcome to Introduction to Bioinformatics 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

You: A typical grad student

Your object of study: Cyanobacteria

How do they do it? Critical position in food web CO 2 sugar N 2 ammonia H 2 O electrons Your object of study: Cyanobacteria

heterocysts Matveyev and Elhai (unpublished) CO 2 sucrose N2N2 N 2 fixation in cyanobacteria O2O2

heterocysts Matveyev and Elhai (unpublished) CO 2 sucrose N2N2 NH 3 N 2 fixation in cyanobacteria O2O2

-NH 3 Differentiation in cyanobacteria Heterocysts ? ? ? ? ?

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

How do bacteria respond to the environment? From gene to protein DNA RNA protein RNA Pol P

High N Low N How do cyanobacteria respond to NH 3 ? From gene to proteinNH 3 α-ketoglutarate glutamine DNA binding protein, NtcA RNA Pol Binding site P DNA No RNA

High N Low N How do cyanobacteria respond to NH 3 ? From gene to proteinNH 3 α-ketoglutarate glutamine DNA binding protein, NtcA RNA Pol Binding site P DNA No RNA

How do cyanobacteria respond to NH 3 ? From gene to protein DNA Low N RNA Pol NtcA Binding site P RNA protein α-ketoglutarate

-NH 3 Differentiation in cyanobacteria Heterocysts ? ? ? ? ?

-NH 3 Differentiation in cyanobacteria Heterocysts ? ? ? Activates NtcA (Nitrogen Control)

Differentiation in cyanobacteria What DNA site does NtcA bind to? RNA Pol NtcA Binding site P

Differentiation in cyanobacteria What DNA site does NtcA bind to?

Herrero et al (2001) J Bacteriol 183: mRNA …(20-24)…TAnnnT GTA…(8)…TAC RNA Pol NtcA Binding site P

HetR Genes needed for differentiation Master regulator Differentiation in cyanobacteria Integration of signals through HetR Level of PatS Level of HetN Position in cell cycle NtcA -N ??? Strategy PCR out hetQ Random mutagenesis Look for effects on HetR expression/activity HetQ

cctatctccgccctatggcgatttgggcaatatatttgatgattggttag...hypothetical ttgtcagttgtcagacgtagtagcgcgtctagtctaatgtgttgttatat protein tatttgctactagaaatgaggagagggttatttttctcactgcttcccaa ttctatgagaatataaaattttccttaagtttctcatggcaataatggaa aaaaccgaccattctgatgaataagtccggttttttccaaaaaatatttt tgctttttcgctttatttatctatatttccaagttttagtacatcggtga ggggtgacaactatcttgccaatattgtcgttattgttaggttgctatcg gaaaaaatctgtaacatgagatacacaatagcatttatatttgctttagt atctctctcttgggtgggattctgcctgcaatttaaaaaccagtgttaac aattttcggctttattttccgggagttaaatcaaccaagggaaaatgtaa ctaatgtttaaatatcttcggatacacacaaagtaaaaccaatttttaca gatgtcgatgttgctcacattttttagaaatattactaaattaaaaatgt tattaaatttatgttcatagagaaccttttccaaataaaaaaataatttt cctgatgttttaagaaaattactgttgttataaattaaaggtgattcaac aaaatatagatagttctttcaataactatctacttttaccattaagtgaa cttactcatgaataatcaacaggaattaaaaataaagttcatgaatactg gttaaagattcagtaaagtttgaggaaataccggaataaatttccaccca aatatgattttttaaaagatacattggcagtacattaaaatgccgatgtt agataaatttgccttcatagctgttatctatttgctcagaactaagccaa gagtttacacaccaaacagaaattaaactatgaatccctcttcgtcgtta hetQ... Differentiation in cyanobacteria Find 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...

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

ttctatgagaatataaaattttccttaagtttct aaaaccgaccattctgatgaataagtccggtttt tgctttttcgctttatttatctatatttccaagt ggggtgacaactatcttgccaatattgtcgttat gaaaaaatctGTAacatgagaTACacaatagcat ttatatttgcttTAgtaTctctctcttgggtggg Differentiation in cyanobacteria GTA…(8)…TAC NtcA binding site …(20-24)…TAnnnT Promoter

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

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

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

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

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

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

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

Regulatory Protein and their Binding Sites Palindromic sequences What is it? What about with DNA? GCTATCG Backwards = forwards ROTATOR TTAATGTGAGTTAGCTCACTCATT AATTACACTCAATCGAGTGAGTAA DNA is double stranded

Regulatory Protein and their Binding Sites Palindromic sequences What is it? What about with DNA? GCTATCG Backwards = forwards ROTATOR DNA is redundant TTAATGTGAGTTAGCTCACTCATT AATTACACTCAATCGAGTGAGTAA DNA is double stranded

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

Regulatory Protein and their Binding Sites Palindromic sequences TTAATGTGAGTTAGCTCACTCATT AATTACACTCAATCGAGTGAGTAA 5’- -3’ 3’- -5’ TA T G GC AT GC TA GC TTAAT TCATT AATTA AGTAA CG TA CG AT CG G T AT DNA: cruciform RNA: stem/loop

Regulatory Protein and their Binding Sites Palindromic sequences TTAATGTGAGTTAGCTCACTCATT AATTACACTCAATCGAGTGAGTAA 5’- -3’ 3’- -5’ tRNA UA U G GC AU GC UA GC UUAAU UCAUU DNA: cruciform RNA: stem/loop

Regulatory Protein and their Binding Sites Palindromic sequences recognizes GTGAGTT NNNNNNNNNNNNNNNNNNNNNNNNNNNN TTAATGTGAGTTAGCTCACTCATT AATGAGTGAGCTAACTCACATTAA

Regulatory Protein and their Binding Sites Palindromic sequences NNNNNNNNNNNNNNNNNNNNNNNNNNNN TTAATGTGAGTTAGCTCACTCATT AATGAGTGAGCTAACTCACATTAA

Regulatory Protein and their Binding Sites Palindromic sequences NNNNNNNNNNNNNNNNNNNNNNNNNNNN TTAATGTGAGTTAGCTCACTCATT AATGAGTGAGCTAACTCACATTAA

Regulatory Protein and their Binding Sites Palindromic sequences NNNNNNNNNNNNNNNNNNNNNNNNNNNN TTAATGTGAGTTAGCTCACTCATT AATGAGTGAGCTAACTCACATTAA

Regulatory Protein and their Binding Sites Palindromic sequences NNNNNNNNNNNNNNNNNNNNNNNNNNNN TTAATGTGAGTTAGCTCACTCATT AATGAGTGAGCTAACTCACATTAA

recognizes GTGAGTT Regulatory Protein and their Binding Sites Palindromic sequences NNNNNNNNNNNNNNNNNNNNNNNNNNNN TTAATGTGAGTTAGCTCACTCATT AATGAGTGAGCTAACTCACATTAA

Regulatory Protein and their Binding Sites Palindromic sequences NNNNNNNNNNNNNNN NNNNNNNNNNNNN TTAATGTGAGTTAGCTCACTCATT AATGAGTGAGCTAACTCACATTAA

Regulatory Protein and their Binding Sites Palindromic sequences NNNNNNNNNNNNNNN NNNNNNNNNNNNN TTAATGTGAGTTAGCTCACTCATT AATGAGTGAGCTAACTCACATTAA

Palindromes: Serve as binding sites for dimeric protein Regulatory Protein and their Binding Sites Palindromic sequences NNNNNNNNNNNNNNN NNNNNNNNNNNNN TTAATGTGAGTTAGCTCACTCATT AATGAGTGAGCTAACTCACATTAA

Regulatory Protein and their Binding Sites SQ7: Is the binding site of NtcA palindromic? GTA..(8).. TAC 5’-GTA..(8).. TAC-3’ 3’-CAT..(8).. ATG -5’

Regulatory Protein and their Binding Sites SQ7: How does NtcA bind to its binding site? GTA..(8).. TAC 5’-GTA..(8).. TAC-3’ 3’-CAT..(8).. ATG -5’

Regulatory Protein and their Binding Sites SQ8: What if NtcA site were attached to lacZ? GTA..(8).. TAC 5’-GTA..(8).. TACNNNNNNNNNNTANNNTNNNNNNNNNNNNNNNNNNNNNNNNNNNNATGNNNNNNNNNNNNNNNN 3’-CAT..(8).. ATGNNNNNNNNNNATNNNANNNNNNNNNNNNNNNNNNNNNNNNNNNNTACNNNNNNNNNNNNNNNN hetQ NtcA N RNA Polymerase

Regulatory Protein and their Binding Sites SQ8: What if NtcA site were attached to lacZ?

Regulatory Protein and their Binding Sites SQ8: What if NtcA site were attached to lacZ? GTA..(8).. TAC 5’-GTA..(8).. TACNNNNNNNNNNTANNNTNNNNNNNNNNNNNNNNNNNNNNNNNNNNATGNNNNNNNNNNNNNNNN 3’-CAT..(8).. ATGNNNNNNNNNNATNNNANNNNNNNNNNNNNNNNNNNNNNNNNNNNTACNNNNNNNNNNNNNNNN hetQ NtcA GTA..(8).. TAC 5’-GTGAGTTAGCTCACNNNNNNNNNNTANNNTNNNNNNNNNNNNNNNNNNNNNNNNNNNNATGNNNNNNNNNNNNNNNN 3’-CACTCAATCGAGTGNNNNNNNNNATNNNANNNNNNNNNNNNNNNNNNNNNNNNNNNNTACNNNNNNNNNNNNNNNN lacZ Crp RNA Polymerase Operator NC

Regulatory Protein and their Binding Sites SQ8: What if NtcA site were attached to lacZ? GTA..(8).. TAC 5’-GTA..(8).. TACNNNNNNNNNNTANNNTNNNNNNNNNNNNNNNNNNNNNNNNNNNNATGNNNNNNNNNNNNNNNN 3’-CAT..(8).. ATGNNNNNNNNNNATNNNANNNNNNNNNNNNNNNNNNNNNNNNNNNNTACNNNNNNNNNNNNNNNN hetQ NtcA GTA..(8).. TAC 5’-GTA..(8).. TACNNNNNNNNNNTANNNTNNNNNNNNNNNNNNNNNNNNNNNNNNNNATGNNNNNNNNNNNNNNNN 3’-CAT..(8).. ATGNNNNNNNNNNATNNNANNNNNNNNNNNNNNNNNNNNNNNNNNNNTACNNNNNNNNNNNNNNNN lacZ RNA Polymerase Operator N

Problem Set 2M, Problem 2