BBSI Research Simulation News

BBSI Research SimulationNews

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BBSI Research SimulationPSSMs and Search for Repeats in DNA

Application of PSSMs

• Regulatory protein and their binding sites• Palindromic DNA and its significance

• How to find protein binding sites: Meme

• PSSMs to find beginning of genes

• Repeated sequences and location of protein binding sites Li et al (2002)

Regulatory Protein and their Binding Sites

GTA ..(8).. TAC

5’-GTGAGTTAGCTCACNNNNNNNNNNTANNNTNNNNNNNNNNNNNNNNNNNNNNNNNNNNATGNNNNNNNNNNNNNNNN3’-CACTCAATCGAGTGNNNNNNNNNNATNNNANNNNNNNNNNNNNNNNNNNNNNNNNNNNTACNNNNNNNNNNNNNNNN

lacZ

Crp

RNA Polymerase

Operator

C

Presence of CRP sites Regulation by carbon source

Presence of X sites Regulation by Y


5’-GTGAGTTAGCTCACNNNNNNNNNNTANNNTNNNNNNNNNNNNNNNNNNNNNNNNNNNNATGNNNNNNNNNNNNNNNN3’-CACTCAATCGAGTGNNNNNNNNNNATNNNANNNNNNNNNNNNNNNNNNNNNNNNNNNNTACNNNNNNNNNNNNNNNN

Regulatory Protein and their Binding SitesPalindromic sequences

TTAATGTGAGTTAGCTCACTCATTAATTACACTCAATCGAGTGAGTAA

NNNNNNNNNNNNNNNNNNNNNNNNNNNNNN

NNNNNNNNNNNNNNNNNNNNNNNNNN

recognizes GTGAGTT





recognizes GTGAGTT






Palindromic sequencesTTAATGTGAGTTAGCTCACTCATT

AATTACACTCAATCGAGTGAGTAA

NNNNNNNNNNNNNNN

NNNNNNNNNNNNNNN



Palindromic sequences

TTAATGTGAGTTAGCTCACTCATT


NNNNNNNNNNNNNNN

NNNNNNNNNNNNNNN

NNNNNNNNNNNNN

NNNNNNNNNNNNN

Regulatory P

rotein and their Binding Sites

Palindromic sequences



NNNNNNNNNNNNNNN

NNNNNNNNNNNNNNN

NNNNNNNNNNNNN

NNNNNNNNNNNNN





recognizes GTGAGTT


Palindromic sequencesTTAA

TGTGAGTT

AGCTCACT

CATT

AATTACAC

TCAATCGA

GTGAGTAA

NNNNNNNN

NNNNNNN

NNNNNNNN

NNNNNNN

NNNNNNNN

NNNNN

NNNNNNNN

NNNNN

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



5’- -3’3’- -5’

tRNA

TAT GGCATGCTAGC

TTAAT TCATTAATTA AGTAA

CGTACGATCGG TAT

DNA: cruciform

RNA: stem/loop



5’- -3’3’- -5’

TAT GGCATGCTAGC


CGTACGATCGG TAT


Function of palindromeRNA secondary structure?

Binding site for dimeric protein?

How to tell?



5’- -3’3’- -5’

TAT GGCATACTAGC


CGTACGATCGG TAT




How to tell?



5’- -3’3’- -5’

TAT GGCATA CTAGC


CGTACGATCGG TAT




How to tell?



5’- -3’3’- -5’

TAT GGCATA TTAGC


CGTACGATCGG TAT




How to tell?



5’- -3’3’- -5’

TAT GGCATATTAGC


CGTACGATCGG TAT




How to tell?



5’- -3’3’- -5’

TAT GGCATGCTAGC


CGTACGATCGG TAT




How to tell?



5’- -3’3’- -5’

TAT GGCATGCTAGC


CGTACGATCGG TAT

TTAATGTAAGTTAGCTCACTCATTAATTACACTCAATCGAGTGAGTAA



How to tell?



5’- -3’3’- -5’

TAT GGCATGCTAGC


CGTACGATCGG TAT

TTAATGTAAGTTAGCTCACTCATTAATTACATTCAATCGAGTGAGTAA



How to tell?



5’- -3’3’- -5’

TAT GGCATGCTAGC


CGTACGATCGG TAT




How to tell?



5’- -3’3’- -5’

TAT GGCATGCTAGC


CGTACGATCGG TAT


How to tell?Compensatory mutations: RNAUncorrelated mutations: protein

Count all in certain class (Li et al, 2000) Guess a pattern and improve (Meme, Gibbs sampler)

snRNA U1 (pU1-6) AGGTATATGGAGCTGTGACAGGGCAGAAGTGTGTGAAGTChistone H1t GCCCTACCCTATATAAGGCCCCGAGGCCGCCCGGGTGTTTHMG-14 CGGCCGGCGGGGAGGGGGAGCCCGCGGCCGGGGACGCGGGTP1 GCCAAGGCCTTAAATACCCAGACTCCTGCCCCCGGGCCTTprotamine P1 CCCTGGCATCTATAACAGGCCGCAGAGCTGGCCCCTGACTnucleolin GCAGGCTCAGTCTTTCGCCTCAGTCTCGAGCTCTCGCTGGsnRNP E TGCCGCCGCGTGACCTTCACACTTCCGCTTCCGGTTCTTTrp S14 GACACGGAAGTGACCCCCGTCGCTCCGCCCTCTCCCACTCrp S17 TGGCCTAAGCTTTAACAGGCTTCGCCTGTGCTTCCTGTTTribosomal p. S19 ACCCTACGCCCGACTTGTGCGCCCGGGAAACCCCGTCGTTa'-tubulin ba'1 GGTCTGGGCGTCCCGGCTGGGCCCCGTGTCTGTGCGCACGb'-tubulin b'2 GGGAGGGTATATAAGCGTTGGCGGACGGTCGGTTGTAGCAa'-actin skel-m. CCGCGGGCTATATAAAACCTGAGCAGAGGGACAAGCGGCCa'-cardiac actin TCAGCGTTCTATAAAGCGGCCCTCCTGGAGCCAGCCACCCb'-actin CGCGGCGGCGCCCTATAAAACCCAGCGGCGCGACGCGCCA

Human sequences 5’ to transcriptional start

Regulatory Protein and their Binding SitesHow to find them?

Step 1. Arbitrarily choose candidate pattern from a sequence

Step 2. Find best matches to pattern in all sequences

Step 3. Construct position-dependent frequency table based on matches

Step 4. Calculate relative probability of matches from frequency table

ACAGGGCAGAACCCGGGTGTTTCCGGGGACGCGCCCCCGGGCCTCCGCAGAGCTG

A 0.208 0.292 0.000 0.999 0.000 0.999 0.811 0.905 0.575 0.321 0.151 0.264T 0.160 0.217 0.867 0.000 0.999 0.000 0.189 0.000 0.208 0.057 0.104 0.113C 0.283 0.236 0.132 0.000 0.000 0.000 0.000 0.000 0.000 0.151 0.330 0.283G 0.349 0.255 0.000 0.000 0.000 0.000 0.000 0.95 0.217 0.472 0.415 0.340

Regulatory Protein and their Binding SitesHow Meme finds them

snRNA U1 (pU1-6) AGGTATATGGAGCTGTGACAGGGCAGAAGTGTGTGAAGTChistone H1t GCCCTACCCTATATAAGGCCCCGAGGCCGCCCGGGTGTTTHMG-14 CGGCCGGCGGGGAGGGGGAGCCCGCGGCCGGGGACGCGGGTP1 GCCAAGGCCTTAAATACCCAGACTCCTGCCCCCGGGCCTTprotamine P1 CCCTGGCATCTATAACAGGCCGCAGAGCTGGCCCCTGACT

Human sequences 5’ to transcriptional start

How do pattern finders work?






Step 5. Move around to find local maximum










Step 6. If probability score high, remember pattern and score







Step 7. Repeat Steps 1 - 5



Step 6. If probability score high, remember pattern and score

• You’ve found a gene related to Purple Tongue Syndrome

• BlastP: Encoded protein related to cAMP-binding proteins

• Are the similarities trivial? Related to cAMP binding?

• Does your protein contain cAMP-binding site?

• What IS a cAMP-binding site?

Task

1. Determine what is a cAMP-binding site

2. Determine if your protein has one


1. Collect sequences of known cAMP-binding proteins

2. Run Meme, a pattern-finding programAsk it to find any significant motifs

3. Rerun Meme. Demand that every protein has identified motifs

4. Run Pfam over known sequence to check

Do it

Strategy


aceB ACTATGGAGCATCTGCACATGAAAACCatpI ACCTCGAAGGGAGCAGGAGTGAAAAACbioB ACGTTTTGGAGAAGCCCCATGGCTCACglnA ATCCAGGAGAGTTAAAGTATGTCCGCTglnH TAGAAAAAAGGAAATGCTATGAAGTCTlacZ TTCACACAGGAAACAGCTATGACCATGrpsJ AATTGGAGCTCTGGTCTCATGCAGAACserC GCAACGTGGTGAGGGGAAATGGCTCAAsucA GATGCTTAAGGGATCACGATGCAGAACtrpE CAAAATTAGAGAATAACAATGCAAACA

PSSMs in actionIdentification of beginning of gene

Experimentally proven

start sites

unknown


Experimentally proven

start sites

unknownaceB ACTATGGAGCATCTGCACATGAAAACCatpI ACCTCGAAGGGAGCAGGAGTGAAAAACbioB ACGTTTTGGAGAAGCCCCATGGCTCACglnA ATCCAGGAGAGTTAAAGTATGTCCGCTglnH TAGAAAAAAGGAAATGCTATGAAGTCTlacZ TTCACACAGGAAACAGCTATGACCATGrpsJ AATTGGAGCTCTGGTCTCATGCAGAACserC GCAACGTGGTGAGGGGAAATGGCTCAAsucA GATGCTTAAGGGATCACGATGCAGAACtrpE CAAAATTAGAGAATAACAATGCAAACA

aceB ACCACATAACTATGGAGCATCTGCACATGAAAACCatpI ACCTCGAAGGGAGCAG.....GAGTGAAAAACbioB ACGTTTTGGAGAAGC...CCCATGGCTCACglnA ATCCAGGAGAGTTA.AAGTATGTCCGCTglnH TAGAAAAAAGGAAATG.....CTATGAAGTCTlacZ TTCACACAGGAAACAG....CTATGACCATGrpsJ AATTGGAGCTCTGGTCTCATGCAGAACserC GCAACGTGGTGAGGG...GAAATGGCTCAAsucA GATGCTTAAGGGATCA....CGATGCAGAACtrpE CAAAATTAGAGAATA...ACAATGCAAACA

ACGT


aceB ACCACATAACTATGGAGCATCT.GCACATGAAAACCatpI ACCTCGAAGGGAGCAG.....GAGTGAAAAACbioB ACGTTTTGGAGAAGC...CCCATGGCTCACglnA ATCCAGGAGAGTTA.AAGTATGTCCGCTglnH TAGAAAAAAGGAAATG.....CTATGAAGTCTlacZ TTCACACAGGAAACAG....CTATGACCATGrpsJ AATTGGAGCTCTGGTCTCATGCAGAACserC GCAACGTGGTGAGGG...GAAATGGCTCAAsucA GATGCTTAAGGGATCA....CGATGCAGAACtrpE CAAAATTAGAGAATA...ACAATGCAAACA

ACGT


PSSMs in actionAlgorithm to find binding sites (Li et al)

Li et al (2002)Algorithm

Calculation of probability by Poisson equation

Dimer occurred n times. How likely is that?

Frequency of GTGAGTT = f1

Frequency of AACTCAC = f2

How likely is it to find: GTGAGTTAACTCAC

Frequency of joint occurrence = f1 · f2 = f12



Probability of n occurrences of dimer =

f12 · f12 · f12 · … · (1-f12) · (1-f12) · (1-f12) · …n times N - n times

NCn

N !

n! · (N – n)!·




(f12)n · (1-f12)(N-n)N !

n! · (N – n)!·

Expected number = m = f12 · N

f12 = m / N

(m/N)n · (1-m/N)(N-n)N !

n! · (N – n)!·




(m/N)n · (1-m/N)(N-n)N !

n! · (N – n)!·

(m)n · (1-m/N)NN !

n! · (N – n)!·

(N)n · (1-m/N)n

(m)n · (1-m/N)NN !

n! · (N – n)!·

(N)n · (1 )n

(m)n · e-mN !

n! · (N – n)!·

(N)n · (1 )n




(m)n · e-mN !

n! · (N – n)!·

(N)n · (1 )n

(m)n · e-mN · (N-1) · (N – 2) · … (N–n+1)

n! (N)n · (1 )n

·

(m)n · e-m

n!

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