welcome to introduction to bioinformatics scenario 2: simulation finding biologically important...
TRANSCRIPT
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Welcome toIntroduction to Bioinformatics
Scenario 2: Simulation
Finding biologically important sites in DNAHow to avoid being fooled by imposters?
• Scenario• Gene regulation
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Scenario 2
Finding biologically important sites in DNA
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You: A typical grad student
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You: A typical grad student
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Your object of study: Cyanobacteria
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How do they do it?
Critical position in food web
CO2 sugarN2 ammoniaH2O electrons
Your object of study: Cyanobacteria
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heterocysts
Matveyev and Elhai (unpublished)
CO2
sucroseN2
N2 fixation in cyanobacteria
O2
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heterocysts
Matveyev and Elhai (unpublished)
CO2
sucroseN2
NH3
NH3
N2 fixation in cyanobacteria
O2
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-NH3
Differentiation in cyanobacteria
Heterocysts
? ? ? ? ?
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DNA
RNA protein
How do bacteria respond to the environment?From gene to protein
Response to environment
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How do bacteria respond to the environment?From gene to protein
DNA
RNA
protein
RNAPol
P
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High NLow N
How do cyanobacteria respond to NH3?
From gene to protein
NH3
α-ketoglutarateglutamine
DNA binding protein, NtcA
RNA
Pol
Binding site
P
DNA
No RNA
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High NLow N
How do cyanobacteria respond to NH3?
From gene to protein
NH3
α-ketoglutarateglutamine
DNA binding protein, NtcA
RNA
Pol
Binding site
P
DNA
No RNA
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How do cyanobacteria respond to NH3?
From gene to protein
DNA
Low N
RNAPolNtcA
Binding site
P
RNA
protein
α-ketoglutarate
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-NH3
Differentiation in cyanobacteria
Heterocysts
? ? ? ? ?
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-NH3
Differentiation in cyanobacteria
Heterocysts
? ? ?
Activates NtcA (Nitrogen Control)
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Differentiation in cyanobacteriaWhat DNA site does NtcA bind to?
RNAPolNtcA
Binding site
P
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Differentiation in cyanobacteriaWhat DNA site does NtcA bind to?
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Differentiation in cyanobacteriaWhat DNA site does NtcA bind to?
Herrero et al (2001) J Bacteriol 183:411-425
mRNA
…(20-24)…TAnnnTGTA…(8)…TAC
RNAPolNtcA
Binding site
P
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HetRGenes
needed for differentiation
Master regulator
Differentiation in cyanobacteriaIntegration of signals through HetR
Level of PatS
Level of HetN
Position in cell cycle
NtcA -N???
StrategyPCR out hetQRandom mutagenesisLook for effects on HetR expression/activity
HetQ
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cctatctccgccctatggcgatttgggcaatatatttgatgattggttag ...hypotheticalttgtcagttgtcagacgtagtagcgcgtctagtctaatgtgttgttatat proteintatttgctactagaaatgaggagagggttatttttctcactgcttcccaattctatgagaatataaaattttccttaagtttctcatggcaataatggaaaaaaccgaccattctgatgaataagtccggttttttccaaaaaatatttttgctttttcgctttatttatctatatttccaagttttagtacatcggtgaggggtgacaactatcttgccaatattgtcgttattgttaggttgctatcggaaaaaatctgtaacatgagatacacaatagcatttatatttgctttagtatctctctcttgggtgggattctgcctgcaatttaaaaaccagtgttaacaattttcggctttattttccgggagttaaatcaaccaagggaaaatgtaactaatgtttaaatatcttcggatacacacaaagtaaaaccaatttttacagatgtcgatgttgctcacattttttagaaatattactaaattaaaaatgttattaaatttatgttcatagagaaccttttccaaataaaaaaataattttcctgatgttttaagaaaattactgttgttataaattaaaggtgattcaacaaaatatagatagttctttcaataactatctacttttaccattaagtgaacttactcatgaataatcaacaggaattaaaaataaagttcatgaatactggttaaagattcagtaaagtttgaggaaataccggaataaatttccacccaaatatgattttttaaaagatacattggcagtacattaaaatgccgatgtt agataaatttgccttcatagctgttatctatttgctcagaactaagccaagagtttacacaccaaacagaaattaaactatgaatccctcttcgtcgtta hetQ...
Differentiation in cyanobacteriaFind primers to PCR out hetQ
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Differentiation in cyanobacteriaFind primers to PCR out hetQ
cctatctccgccctatggcgatttgggcaatatatttgatgattggttag ...hypotheticalttgtcagttgtcagacgtagtagcgcgtctagtctaatgtgttgttatat proteintatttgctactagaaatgaggagagggttatttttctcactgcttcccaattctatgagaatataaaattttccttaagtttctcatggcaataatggaaaaaaccgaccattctgatgaataagtccggttttttccaaaaaatatttttgctttttcgctttatttatctatatttccaagttttagtacatcggtgaggggtgacaactatcttgccaatattgtcgttattgttaggttgctatcggaaaaaatctgtaacatgagatacacaatagcatttatatttgctttagtatctctctcttgggtgggattctgcctgcaatttaaaaaccagtgttaacaattttcggctttattttccgggagttaaatcaaccaagggaaaatgtaactaatgtttaaatatcttcggatacacacaaagtaaaaccaatttttacagatgtcgatgttgctcacattttttagaaatattactaaattaaaaatgttattaaatttatgttcatagagaaccttttccaaataaaaaaataattttcctgatgttttaagaaaattactgttgttataaattaaaggtgattcaacaaaatatagatagttctttcaataactatctacttttaccattaagtgaacttactcatgaataatcaacaggaattaaaaataaagttcatgaatactggttaaagattcagtaaagtttgaggaaataccggaataaatttccacccaaatatgattttttaaaagatacattggcagtacattaaaatgccgatgtt agataaatttgccttcatagctgttatctatttgctcagaactaagccaagagtttacacaccaaacagaaattaaactatgaatccctcttcgtcgtta hetQ...
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ttgtcagttgtcagacgtagtagcgcgtctagtctaatgtgttgttatattatttgctactagaaatgaggagagggttatttttctcactgcttcccaattctatgagaatataaaattttccttaagtttctcatggcaataatggaaaaaaccgaccattctgatgaataagtccggttttttccaaaaaatatttttgctttttcgctttatttatctatatttccaagttttagtacatcggtgaggggtgacaactatcttgccaatattgtcgttattgttaggttgctatcggaaaaaatcTGTAacatgagaTACAcaatagcatttatatttgctttagtatctctctcttgggtgggattctgcctgcaatttaaaaaccagtgttaacaattttcggctttattttccgggagttaaatcaaccaagggaaaatgtaactaatgtttaaatatcttcggatacacacaaagtaaaaccaatttttacagatgtcgatgttgctcacattttttagaaatattactaaattaaaaatgttattaaatttatgttcatagagaaccttttccaaataaaaaaataattttcctgatgttttaagaaaattactgttgttataaattaaaggtgattcaacaaaatatagatagttctttcaataactatctacttttaccattaagtgaacttactcatgaataatcaacaggaattaaaaataaagttcatgaatactggttaaagattcagtaaagtttgaggaaataccggaataaatttccacccaaatatgattttttaaaagatacattggcagtacattaaaatgccgatgtt agataaatttgccttcatagctgttatctatttgctcagaactaagccaagagtttacacaccaaacagaaattaaactatgaatccctcttcgtcgtta hetC...
Differentiation in cyanobacteriaFind primers to PCR out hetC
GTA…(8)…TAC
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ttctatgagaatataaaattttccttaagtttctaaaaccgaccattctgatgaataagtccggtttttgctttttcgctttatttatctatatttccaagtggggtgacaactatcttgccaatattgtcgttatgaaaaaatctGTAacatgagaTACacaatagcatttatatttgcttTAgtaTctctctcttgggtggg
Differentiation in cyanobacteria
GTA…(8)…TAC NtcA binding site
…(20-24)…TAnnnTPromoter
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Level of PatS
Level of HetN
Position in cell cycle
NtcA
HetRGenes
needed for differentiation
-N
Master regulator
Differentiation in cyanobacteriaIntegration of signals through HetR
???
HetQ
Stockholm
??????
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How to proceed?
Choice #1• Publish• Grant proposals• Build a career
Likely result• Reviewers trash MS: too speculative
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How to proceed?
Choice #2• Forget about it• Back to PCR
Likely result• Sometimes miss spectacular finding
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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.
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How to proceed?
Choice #4• Determine whether site is likely to be real
How? N! . . . a! (N-a)!
• High school math approach
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How to proceed?
Choice #4• Determine whether site is likely to be real
How? BIOINFORMATICS• Simulation• Exhaustive pattern search
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Regulatory Protein and their Binding SitesWhat 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)
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Regulatory Protein and their Binding SitesPalindromic sequences
What is it?
What about with DNA? GCTATCG
Backwards = forwards ROTATOR
TTAATGTGAGTTAGCTCACTCATTAATTACACTCAATCGAGTGAGTAA
• DNA is double stranded
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Regulatory Protein and their Binding SitesPalindromic sequences
What is it?
What about with DNA? GCTATCG
Backwards = forwards ROTATOR
• DNA is redundant
TTAATGTGAGTTAGCTCACTCATTAATTACACTCAATCGAGTGAGTAA
• DNA is double stranded
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Regulatory Protein and their Binding SitesPalindromic sequences
What is it?
What about with DNA? GCTATCG
Backwards = forwards ROTATOR
• DNA is redundant
TTAATGTGAGTTAGCTCACTCATTAATTACACTCAATCGAGTGAGTAA
• DNA is double stranded
TTAATGTGAGTTAGCTCACTCATTAATGAGTGAGCTAACTCACATTAA
• DNA has direction (read 5’->3’)
5’- -3’3’- -5’
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Regulatory Protein and their Binding SitesPalindromic sequences
TTAATGTGAGTTAGCTCACTCATTAATTACACTCAATCGAGTGAGTAA
5’- -3’3’- -5’
TAT GGCATGCTAGC
TTAAT TCATTAATTA AGTAA
CGTACGATCGG TAT
DNA: cruciform
RNA: stem/loop
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Regulatory Protein and their Binding SitesPalindromic sequences
TTAATGTGAGTTAGCTCACTCATTAATTACACTCAATCGAGTGAGTAA
5’- -3’3’- -5’
tRNA
UAU GGCAUGCUAGC
UUAAU UCAUU
DNA: cruciform
RNA: stem/loop
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Regulatory Protein and their Binding SitesPalindromic sequences
recognizes GTGAGTT
NNNNNNNNNNNNNNNNNNNNNNNNNNNNNN
NNNNNNNNNNNNNNNNNNNNNNNNNN
TTAATGTGAGTTAGCTCACTCATT AATGAGTGAGCTAACTCACATTAA
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Regulatory Protein and their Binding SitesPalindromic sequences
NNNNNNNNNNNNNNNNNNNNNNNNNNNNNN
NNNNNNNNNNNNNNNNNNNNNNNNNN
TTAATGTGAGTTAGCTCACTCATT AATGAGTGAGCTAACTCACATTAA
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Regulatory Protein and their Binding Sites
Palindromic sequences
NNNNNNNNNNNNNNN
NNNNNNNNNNNNNNN
NNNNNNNNNNNNNNNNNNNNNNNNNN
TTAATGTGAGTTAGCTCACTCATT AATGAGTG
AGCTAACT
CACATTAA
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Regulatory Protein and their Binding Sites
Palindromic sequences
NNNNNNNNNNNNNNN
NNNNNNNNNNNNNNN
NNNNNNNNNNNNN
NNNNNNNNNNNNN
TTAATGTGAGTTAGCTCACTCATT
AATGAGTGAGCTAACTCACATTAA
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Regulatory P
rotein and their Binding Sites
Palindromic sequences
NNNNNNNNNNNNNNN
NNNNNNNNNNNNNNN
NNNNNNNNNNNNN
NNNNNNNNNNNNN
TTAATGTGAGTTAGCTCACTCATT
AATGAGTGAGCTAACTCACATTAA
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recognizes GTGAGTT
Regulatory Protein and their Binding Sites
Palindromic sequences
NNNNNNNNNNNNNNNNNNNNNNNNNNNNNN
NNNNNNNNNNNNNNNNNNNNNNNNNN
TTAATGTGAGTTAGCTCACTCATTAATGAGTGAGCTAACTCACATTAA
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Regulatory Protein and their Binding Sites
Palindromic sequences
NNNNNNNN
NNNNNNN
NNNNNNNN
NNNNNNN
NNNNNNNN
NNNNN
NNNNNNNN
NNNNN
TTAATGTG
AGTTAGCT
CACTCATT
AATGAGTGAGCTAACTCACATTAA
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Regul
ator
y Pro
tein
and
their
Bin
ding
Site
s
Palin
drom
ic se
quen
ces
NNNNNNNNNNNNNNN
NNNNNNNNNNNNNNN
NNNNNNNNNNNNN
NNNNNNNNNNNNN
TTAATGTGAGTTAGCTCACTCATT
AATGAGTGAGCTAACTCACATTAA
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Palindromes: Serve as binding sites for dimeric protein
Regulatory Protein and their Binding SitesPalindromic sequences
NNNNNNNNNNNNNNNNNNNNNNNNNNNNNN
NNNNNNNNNNNNNNNNNNNNNNNNNN
TTAATGTGAGTTAGCTCACTCATT AATGAGTGAGCTAACTCACATTAA
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Regulatory Protein and their Binding SitesSQ7: Is the binding site of NtcA palindromic?
GTA ..(8).. TAC
GTA ..(8).. TAC
5’-GTA ..(8).. TAC-3’3’-CAT ..(8).. ATG -5’
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Regulatory Protein and their Binding SitesSQ7: How does NtcA bind to its binding site?
GTA ..(8).. TAC
GTA ..(8).. TAC
5’-GTA ..(8).. TAC-3’3’-CAT ..(8).. ATG -5’
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Regulatory Protein and their Binding SitesSQ8: What if NtcA site were attached to lacZ?
GTA ..(8).. TAC
5’-GTA ..(8).. TACNNNNNNNNNNTANNNTNNNNNNNNNNNNNNNNNNNNNNNNNNNNATGNNNNNNNNNNNNNNNN3’-CAT ..(8).. ATGNNNNNNNNNNATNNNANNNNNNNNNNNNNNNNNNNNNNNNNNNNTACNNNNNNNNNNNNNNNN
hetQ
NtcA
N RNA Polymerase
![Page 49: Welcome to Introduction to Bioinformatics Scenario 2: Simulation Finding biologically important sites in DNA How to avoid being fooled by imposters? Scenario](https://reader036.vdocuments.us/reader036/viewer/2022070413/5697bfee1a28abf838cb98d8/html5/thumbnails/49.jpg)
Regulatory Protein and their Binding SitesSQ8: What if NtcA site were attached to lacZ?
![Page 50: Welcome to Introduction to Bioinformatics Scenario 2: Simulation Finding biologically important sites in DNA How to avoid being fooled by imposters? Scenario](https://reader036.vdocuments.us/reader036/viewer/2022070413/5697bfee1a28abf838cb98d8/html5/thumbnails/50.jpg)
Regulatory Protein and their Binding SitesSQ8: What if NtcA site were attached to lacZ?
GTA ..(8).. TAC
5’-GTA ..(8).. TACNNNNNNNNNNTANNNTNNNNNNNNNNNNNNNNNNNNNNNNNNNNATGNNNNNNNNNNNNNNNN3’-CAT ..(8).. ATGNNNNNNNNNNATNNNANNNNNNNNNNNNNNNNNNNNNNNNNNNNTACNNNNNNNNNNNNNNNN
hetQ
NtcA
GTA ..(8).. TAC
5’-GTGAGTTAGCTCACNNNNNNNNNNTANNNTNNNNNNNNNNNNNNNNNNNNNNNNNNNNATGNNNNNNNNNNNNNNNN3’-CACTCAATCGAGTGNNNNNNNNNATNNNANNNNNNNNNNNNNNNNNNNNNNNNNNNNTACNNNNNNNNNNNNNNNN
lacZ
Crp
RNA Polymerase
Operator
N
C
![Page 51: Welcome to Introduction to Bioinformatics Scenario 2: Simulation Finding biologically important sites in DNA How to avoid being fooled by imposters? Scenario](https://reader036.vdocuments.us/reader036/viewer/2022070413/5697bfee1a28abf838cb98d8/html5/thumbnails/51.jpg)
Regulatory Protein and their Binding SitesSQ8: What if NtcA site were attached to lacZ?
GTA ..(8).. TAC
5’-GTA ..(8).. TACNNNNNNNNNNTANNNTNNNNNNNNNNNNNNNNNNNNNNNNNNNNATGNNNNNNNNNNNNNNNN3’-CAT ..(8).. ATGNNNNNNNNNNATNNNANNNNNNNNNNNNNNNNNNNNNNNNNNNNTACNNNNNNNNNNNNNNNN
hetQ
NtcAGTA ..(8).. TAC
5’-GTA ..(8).. TACNNNNNNNNNNTANNNTNNNNNNNNNNNNNNNNNNNNNNNNNNNNATGNNNNNNNNNNNNNNNN3’-CAT ..(8).. ATGNNNNNNNNNNATNNNANNNNNNNNNNNNNNNNNNNNNNNNNNNNTACNNNNNNNNNNNNNNNN
lacZ
RNA Polymerase
Operator
N
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Problem Set 2M, Problem 2