Welcome to Introduction to Bioinformatics Scenario 2: Simulation Finding biologically important sites in DNA How to avoid being fooled by imposters? Scenario

Welcome toIntroduction to Bioinformatics

Scenario 2: Simulation

Finding biologically important sites in DNAHow to avoid being fooled by imposters?

• Scenario• Gene regulation

Scenario 2

Finding biologically important sites in DNA

You: A typical grad student

You: A typical grad student

Your object of study: Cyanobacteria

How do they do it?

Critical position in food web

CO2 sugarN2 ammoniaH2O electrons

Your object of study: Cyanobacteria

heterocysts

Matveyev and Elhai (unpublished)

CO2

sucroseN2

N2 fixation in cyanobacteria

O2

heterocysts

Matveyev and Elhai (unpublished)

CO2

sucroseN2

NH3

NH3

N2 fixation in cyanobacteria

O2

-NH3

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

RNAPol

P

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

High NLow N



NH3

α-ketoglutarateglutamine

DNA binding protein, NtcA

RNA

Pol

Binding site

P

DNA

No RNA



DNA

Low N

RNAPolNtcA

Binding site

P

RNA

protein

α-ketoglutarate

-NH3


Heterocysts

? ? ? ? ?

-NH3


Heterocysts

? ? ?

Activates NtcA (Nitrogen Control)

Differentiation in cyanobacteriaWhat DNA site does NtcA bind to?

RNAPolNtcA

Binding site

P



Herrero et al (2001) J Bacteriol 183:411-425

mRNA

…(20-24)…TAnnnTGTA…(8)…TAC

RNAPolNtcA

Binding site

P

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

cctatctccgccctatggcgatttgggcaatatatttgatgattggttag ...hypotheticalttgtcagttgtcagacgtagtagcgcgtctagtctaatgtgttgttatat proteintatttgctactagaaatgaggagagggttatttttctcactgcttcccaattctatgagaatataaaattttccttaagtttctcatggcaataatggaaaaaaccgaccattctgatgaataagtccggttttttccaaaaaatatttttgctttttcgctttatttatctatatttccaagttttagtacatcggtgaggggtgacaactatcttgccaatattgtcgttattgttaggttgctatcggaaaaaatctgtaacatgagatacacaatagcatttatatttgctttagtatctctctcttgggtgggattctgcctgcaatttaaaaaccagtgttaacaattttcggctttattttccgggagttaaatcaaccaagggaaaatgtaactaatgtttaaatatcttcggatacacacaaagtaaaaccaatttttacagatgtcgatgttgctcacattttttagaaatattactaaattaaaaatgttattaaatttatgttcatagagaaccttttccaaataaaaaaataattttcctgatgttttaagaaaattactgttgttataaattaaaggtgattcaacaaaatatagatagttctttcaataactatctacttttaccattaagtgaacttactcatgaataatcaacaggaattaaaaataaagttcatgaatactggttaaagattcagtaaagtttgaggaaataccggaataaatttccacccaaatatgattttttaaaagatacattggcagtacattaaaatgccgatgtt agataaatttgccttcatagctgttatctatttgctcagaactaagccaagagtttacacaccaaacagaaattaaactatgaatccctcttcgtcgtta hetQ...

Differentiation in cyanobacteriaFind primers to PCR out hetQ

Differentiation in cyanobacteriaFind primers to PCR out hetQ

cctatctccgccctatggcgatttgggcaatatatttgatgattggttag ...hypotheticalttgtcagttgtcagacgtagtagcgcgtctagtctaatgtgttgttatat proteintatttgctactagaaatgaggagagggttatttttctcactgcttcccaattctatgagaatataaaattttccttaagtttctcatggcaataatggaaaaaaccgaccattctgatgaataagtccggttttttccaaaaaatatttttgctttttcgctttatttatctatatttccaagttttagtacatcggtgaggggtgacaactatcttgccaatattgtcgttattgttaggttgctatcggaaaaaatctgtaacatgagatacacaatagcatttatatttgctttagtatctctctcttgggtgggattctgcctgcaatttaaaaaccagtgttaacaattttcggctttattttccgggagttaaatcaaccaagggaaaatgtaactaatgtttaaatatcttcggatacacacaaagtaaaaccaatttttacagatgtcgatgttgctcacattttttagaaatattactaaattaaaaatgttattaaatttatgttcatagagaaccttttccaaataaaaaaataattttcctgatgttttaagaaaattactgttgttataaattaaaggtgattcaacaaaatatagatagttctttcaataactatctacttttaccattaagtgaacttactcatgaataatcaacaggaattaaaaataaagttcatgaatactggttaaagattcagtaaagtttgaggaaataccggaataaatttccacccaaatatgattttttaaaagatacattggcagtacattaaaatgccgatgtt agataaatttgccttcatagctgttatctatttgctcagaactaagccaagagtttacacaccaaacagaaattaaactatgaatccctcttcgtcgtta hetQ...

ttgtcagttgtcagacgtagtagcgcgtctagtctaatgtgttgttatattatttgctactagaaatgaggagagggttatttttctcactgcttcccaattctatgagaatataaaattttccttaagtttctcatggcaataatggaaaaaaccgaccattctgatgaataagtccggttttttccaaaaaatatttttgctttttcgctttatttatctatatttccaagttttagtacatcggtgaggggtgacaactatcttgccaatattgtcgttattgttaggttgctatcggaaaaaatcTGTAacatgagaTACAcaatagcatttatatttgctttagtatctctctcttgggtgggattctgcctgcaatttaaaaaccagtgttaacaattttcggctttattttccgggagttaaatcaaccaagggaaaatgtaactaatgtttaaatatcttcggatacacacaaagtaaaaccaatttttacagatgtcgatgttgctcacattttttagaaatattactaaattaaaaatgttattaaatttatgttcatagagaaccttttccaaataaaaaaataattttcctgatgttttaagaaaattactgttgttataaattaaaggtgattcaacaaaatatagatagttctttcaataactatctacttttaccattaagtgaacttactcatgaataatcaacaggaattaaaaataaagttcatgaatactggttaaagattcagtaaagtttgaggaaataccggaataaatttccacccaaatatgattttttaaaagatacattggcagtacattaaaatgccgatgtt agataaatttgccttcatagctgttatctatttgctcagaactaagccaagagtttacacaccaaacagaaattaaactatgaatccctcttcgtcgtta hetC...

Differentiation in cyanobacteriaFind primers to PCR out hetC

GTA…(8)…TAC

ttctatgagaatataaaattttccttaagtttctaaaaccgaccattctgatgaataagtccggtttttgctttttcgctttatttatctatatttccaagtggggtgacaactatcttgccaatattgtcgttatgaaaaaatctGTAacatgagaTACacaatagcatttatatttgcttTAgtaTctctctcttgggtggg


GTA…(8)…TAC NtcA binding site

…(20-24)…TAnnnTPromoter

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

??????

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 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)

Regulatory Protein and their Binding SitesPalindromic sequences

What is it?

What about with DNA? GCTATCG

Backwards = forwards ROTATOR

TTAATGTGAGTTAGCTCACTCATTAATTACACTCAATCGAGTGAGTAA

• DNA is double stranded


What is it?



• DNA is redundant




What is it?



• DNA is redundant



TTAATGTGAGTTAGCTCACTCATTAATGAGTGAGCTAACTCACATTAA

• DNA has direction (read 5’->3’)

5’- -3’3’- -5’



5’- -3’3’- -5’

TAT GGCATGCTAGC

TTAAT TCATTAATTA AGTAA

CGTACGATCGG TAT

DNA: cruciform

RNA: stem/loop



5’- -3’3’- -5’

tRNA

UAU GGCAUGCUAGC

UUAAU UCAUU

DNA: cruciform

RNA: stem/loop


recognizes GTGAGTT

NNNNNNNNNNNNNNNNNNNNNNNNNNNNNN

NNNNNNNNNNNNNNNNNNNNNNNNNN

TTAATGTGAGTTAGCTCACTCATT AATGAGTGAGCTAACTCACATTAA





Regulatory Protein and their Binding Sites

Palindromic sequences

NNNNNNNNNNNNNNN

NNNNNNNNNNNNNNN


TTAATGTGAGTTAGCTCACTCATT AATGAGTG

AGCTAACT

CACATTAA



NNNNNNNNNNNNNNN

NNNNNNNNNNNNNNN

NNNNNNNNNNNNN

NNNNNNNNNNNNN

TTAATGTGAGTTAGCTCACTCATT

AATGAGTGAGCTAACTCACATTAA

Regulatory P

rotein and their Binding Sites


NNNNNNNNNNNNNNN

NNNNNNNNNNNNNNN

NNNNNNNNNNNNN

NNNNNNNNNNNNN



recognizes GTGAGTT





TTAATGTGAGTTAGCTCACTCATTAATGAGTGAGCTAACTCACATTAA



NNNNNNNN

NNNNNNN

NNNNNNNN

NNNNNNN

NNNNNNNN

NNNNN

NNNNNNNN

NNNNN

TTAATGTG

AGTTAGCT

CACTCATT


Regul

ator

y Pro

tein

and

their

Bin

ding

Site

s

Palin

drom

ic se

quen

ces

NNNNNNNNNNNNNNN

NNNNNNNNNNNNNNN

NNNNNNNNNNNNN

NNNNNNNNNNNNN



Palindromes: Serve as binding sites for dimeric protein





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’

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’

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



GTA ..(8).. TAC


hetQ

NtcA

GTA ..(8).. TAC

5’-GTGAGTTAGCTCACNNNNNNNNNNTANNNTNNNNNNNNNNNNNNNNNNNNNNNNNNNNATGNNNNNNNNNNNNNNNN3’-CACTCAATCGAGTGNNNNNNNNNATNNNANNNNNNNNNNNNNNNNNNNNNNNNNNNNTACNNNNNNNNNNNNNNNN

lacZ

Crp

RNA Polymerase

Operator

N

C


GTA ..(8).. TAC


hetQ

NtcAGTA ..(8).. TAC


lacZ

RNA Polymerase

Operator

N

Problem Set 2M, Problem 2

Documents

Welcome to Introduction to Bioinformatics Scenario 2: Simulation Finding biologically important sites in DNA How to avoid being fooled by imposters? Scenario