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Regulatory Motifs Gene Regulation Promoter Gene -35 -10 RNA polymerase Negative Positive Regulation Rep Regulation Act RNA polymerase 1 What if we believed that a number of genes were regulated by the same transcription factor? TF

  1. Regulatory Motifs Gene Regulation Promoter Gene -35 -10 RNA polymerase Negative Positive Regulation Rep Regulation Act RNA polymerase 1

  2. What if we believed that a number of genes were regulated by the same transcription factor? TF “X” Gene1 Gene2 Gene3 Gene4 Gene5 What if we believed that a number of genes were orthologous? Gene EC Gene HI Gene VC Gene ST Gene PA 2

  3. How do we search upstream sequences for instances of a motif? > Escherichia coli TTGATTCCCTGAATGCCCGCTTAGTGTAACACTACTGTAACCGGCATTTTCTGCTTTTCC TGCCGATATTTTTTCTTATCTACCTCACAAAGGTTAGCAATAACTGCTGGGAAAATTCCG AGTTAGTCGTTATATTCTAT > Haemophilus influenzae ATCTAACGGTACGGATTCTCCAAAGGCCTATGGAATCTTGTAGAATATGAAACGTTCTAA TAAATCATAAAGTTGGAGCAAACGCTCGGCATAAGTAGTAAGTGCCGTGCCTCCGCCATT AGTTACACTAGTGGGACACC > Vibrio cholerae ATTTGTGGCGGTTTTCAAATGCTTGGAGAATGGGTACATGATCCGCTTGGCATTGAAGGT GAGGCTGGCAGCAGCGAAGGTCTGGGGCTGTTTGAACGTTACACGAGTGTAACCGCCGAA CCATGTTGACACGAATTCTG > Salmonella typhi GGTCGGCTTAGACTAGTGTGACCAAAAAGCTTTTGCTGAAGTTTCAGGGTAAGAAGAACC AGCTCCTAGTAAAAAGACTATTGTGACTGAAAAGCGCGTCAGCGCAAAGCCGACCGCACA AAACGCACAAGGAGTTACAG > Pseudomonas aeruginosa ACGCGGCCAGGGTCTTCTCCTGCGAGATCATGCGCGGCGCGCCGCGCATGCCGGCGCCGC TGCTGGAACGCCTCGACCCCAGGGCTACACTAGTTTAACCGGAACGCCGCCAGTGGATCG GCCTGCCCCAGCTATTGCTC If we knew where the motif instances were located in each sequence... > Escherichia coli TTGATTCCCTGAATGCCCGCTTAGT GTAACACTACTGTAAC CGGCATTTTCTGCTTTTCC TGCCGATATTTTTTCTTATCTACCTCACAAAGGTTAGCAATAACTGCTGGGAAAATTCCG AGTTAGTCGTTATATTCTAT > Haemophilus influenzae ATCTAACGGTACGGATTCTCCAAAGGCCTATGGAATCTTGTAGAATATGAAACGTTCTAA TAAATCATAAAGTTGGAGCAAACGCTCGGCATAAGTAGTAAGTGCCGTGCCTCCGCCATT A GTTACACTAGTGGGAC ACC > Vibrio cholerae ATTTGTGGCGGTTTTCAAATGCTTGGAGAATGGGTACATGATCCGCTTGGCATTGAAGGT GAGGCTGGCAGCAGCGAAGGTCTGGGGCTGTTTGAAC GTTACACGAGTGTAAC CGCCGAA CCATGTTGACACGAATTCTG > Salmonella typhi GGTCGG CTTAGACTAGTGTGAC CAAAAAGCTTTTGCTGAAGTTTCAGGGTAAGAAGAACC AGCTCCTAGTAAAAAGACTATTGTGACTGAAAAGCGCGTCAGCGCAAAGCCGACCGCACA AAACGCACAAGGAGTTACAG > Pseudomonas aeruginosa ACGCGGCCAGGGTCTTCTCCTGCGAGATCATGCGCGGCGCGCCGCGCATGCCGGCGCCGC TGCTGGAACGCCTCGACCCCAGG GCTACACTAGTTTAAC CGGAACGCCGCCAGTGGATCG GCCTGCCCCAGCTATTGCTC 3

  4. Then we could determine a motif model! GTAACACTACTGTAAC GTTACACTAGTGGGAC GTTACACGAGTGTAAC CTTAGACTAGTGTGAC GCTACACTAGTTTAAC A 0.0 0.0 .20 1.0 0.0 1.0 0.0 0.0 1.0 0.0 0.0 0.0 0.0 .60 1.0 0.0 C .20 .20 0.0 0.0 .80 0.0 1.0 0.0 0.0 .20 0.0 0.0 0.0 0.0 0.0 1.0 .80 0.0 0.0 0.0 .20 0.0 0.0 .20 0.0 .80 0.0 .80 .20 .40 0.0 0.0 G T 0.0 .80 .80 0.0 0.0 0.0 0.0 .80 0.0 0.0 1.0 .20 .80 0.0 0.0 0.0 G T T A C A C T A G T G T A A C Consensus Sequence But we don’t know the locations of the motif instances... > Escherichia coli TTGATTCCCTGAATGCCCGCTTAGTGTAACACTACTGTAACCGGCATTTTCTGCTTTTCC TGCCGATATTTTTTCTTATCTACCTCACAAAGGTTAGCAATAACTGCTGGGAAAATTCCG AGTTAGTCGTTATATTCTAT > Haemophilus influenzae ATCTAACGGTACGGATTCTCCAAAGGCCTATGGAATCTTGTAGAATATGAAACGTTCTAA TAAATCATAAAGTTGGAGCAAACGCTCGGCATAAGTAGTAAGTGCCGTGCCTCCGCCATT AGTTACACTAGTGGGACACC > Vibrio cholerae ATTTGTGGCGGTTTTCAAATGCTTGGAGAATGGGTACATGATCCGCTTGGCATTGAAGGT GAGGCTGGCAGCAGCGAAGGTCTGGGGCTGTTTGAACGTTACACGAGTGTAACCGCCGAA CCATGTTGACACGAATTCTG > Salmonella typhi GGTCGGCTTAGACTAGTGTGACCAAAAAGCTTTTGCTGAAGTTTCAGGGTAAGAAGAACC AGCTCCTAGTAAAAAGACTATTGTGACTGAAAAGCGCGTCAGCGCAAAGCCGACCGCACA AAACGCACAAGGAGTTACAG > Pseudomonas aeruginosa ACGCGGCCAGGGTCTTCTCCTGCGAGATCATGCGCGGCGCGCCGCGCATGCCGGCGCCGC TGCTGGAACGCCTCGACCCCAGGGCTACACTAGTTTAACCGGAACGCCGCCAGTGGATCG GCCTGCCCCAGCTATTGCTC 4

  5. What if we knew the motif model... A 0.0 0.0 .20 1.0 0.0 1.0 0.0 0.0 1.0 0.0 0.0 0.0 0.0 .60 1.0 0.0 C .20 .20 0.0 0.0 .80 0.0 1.0 0.0 0.0 .20 0.0 0.0 0.0 0.0 0.0 1.0 .80 0.0 0.0 0.0 .20 0.0 0.0 .20 0.0 .80 0.0 .80 .20 .40 0.0 0.0 G T 0.0 .80 .80 0.0 0.0 0.0 0.0 .80 0.0 0.0 1.0 .20 .80 0.0 0.0 0.0 We could determine the location of the motif instance which best matches the model... A 0.0 0.0 .20 1.0 0.0 1.0 0.0 0.0 1.0 0.0 0.0 0.0 0.0 .60 1.0 0.0 C .20 .20 0.0 0.0 .80 0.0 1.0 0.0 0.0 .20 0.0 0.0 0.0 0.0 0.0 1.0 .80 0.0 0.0 0.0 .20 0.0 0.0 .20 0.0 .80 0.0 .80 .20 .40 0.0 0.0 G T 0.0 .80 .80 0.0 0.0 0.0 0.0 .80 0.0 0.0 1.0 .20 .80 0.0 0.0 0.0 Score = 0.0 * .80 * 0.0 * 1.0 * 0.0 * 0.0 * 1.0 * 0.0 * 0.0 * 0.0 * 0.0 * 0.0 * 0.0 * 0.0 * 0.0 * 1.0 Score = 0.01 * .80 * 0.01 * 1.0 * 0.01 * 0.01 * 1.0 * 0.01 * 0.01 * 0.01 * 0.01 * 0.01 * 0.01 * 0.01 * 0.01 * 1.0 Score = 8.0 * 10 -27 TTGATTCCCTGAATGC CCGCTTAGTGTAACACTACTGTAA 5

  6. We could determine the location of the motif instance which best matches the model... A 0.0 0.0 .20 1.0 0.0 1.0 0.0 0.0 1.0 0.0 0.0 0.0 0.0 .60 1.0 0.0 C .20 .20 0.0 0.0 .80 0.0 1.0 0.0 0.0 .20 0.0 0.0 0.0 0.0 0.0 1.0 .80 0.0 0.0 0.0 .20 0.0 0.0 .20 0.0 .80 0.0 .80 .20 .40 0.0 0.0 G T 0.0 .80 .80 0.0 0.0 0.0 0.0 .80 0.0 0.0 1.0 .20 .80 0.0 0.0 0.0 Score = 0.0 * 0.0 * .20 * 0.0 * 0.0 * 0.0 * 1.0 * 0.0 * 0.0 * .80 * 0.0 * 0.0 * .80 * .40 * 0.0 * 1.0 Score = 0.01 * 0.01 * .20 * 0.01 * 0.01 * 0.01 * 1.0 * 0.01 * 0.01 * .80 * 0.01 * 0.01 * .80 * .40 * 0.01 * 1.0 Score = 5.12 * 10 -22 T TGATTCCCTGAATGCC CGCTTAGTGTAACACTACTGTAA We could determine the location of the motif instance which best matches the model... A 0.0 0.0 .20 1.0 0.0 1.0 0.0 0.0 1.0 0.0 0.0 0.0 0.0 .60 1.0 0.0 C .20 .20 0.0 0.0 .80 0.0 1.0 0.0 0.0 .20 0.0 0.0 0.0 0.0 0.0 1.0 .80 0.0 0.0 0.0 .20 0.0 0.0 .20 0.0 .80 0.0 .80 .20 .40 0.0 0.0 G T 0.0 .80 .80 0.0 0.0 0.0 0.0 .80 0.0 0.0 1.0 .20 .80 0.0 0.0 0.0 Score = 7.16 * 10 -28 TTGATTCCCTGAATGCCCGCTTAG TGTAACACTACTGTAA 6

  7. A 0.0 0.0 .20 1.0 0.0 1.0 0.0 0.0 1.0 0.0 0.0 0.0 0.0 .60 1.0 0.0 C .20 .20 0.0 0.0 .80 0.0 1.0 0.0 0.0 .20 0.0 0.0 0.0 0.0 0.0 1.0 .80 0.0 0.0 0.0 .20 0.0 0.0 .20 0.0 .80 0.0 .80 .20 .40 0.0 0.0 G T 0.0 .80 .80 0.0 0.0 0.0 0.0 .80 0.0 0.0 1.0 .20 .80 0.0 0.0 0.0 > Escherichia coli TTGATTCCCTGAATGCCCGCTTAGT GTAACACTACTGTAAC CGGCATTTTCTGCTTTTCC TGCCGATATTTTTTCTTATCTACCTCACAAAGGTTAGCAATAACTGCTGGGAAAATTCCG AGTTAGTCGTTATATTCTAT > Haemophilus influenzae ATCTAACGGTACGGATTCTCCAAAGGCCTATGGAATCTTGTAGAATATGAAACGTTCTAA TAAATCATAAAGTTGGAGCAAACGCTCGGCATAAGTAGTAAGTGCCGTGCCTCCGCCATT A GTTACACTAGTGGGAC ACC > Vibrio cholerae ATTTGTGGCGGTTTTCAAATGCTTGGAGAATGGGTACATGATCCGCTTGGCATTGAAGGT GAGGCTGGCAGCAGCGAAGGTCTGGGGCTGTTTGAAC GTTACACGAGTGTAAC CGCCGAA CCATGTTGACACGAATTCTG > Salmonella typhi GGTCGG CTTAGACTAGTGTGAC CAAAAAGCTTTTGCTGAAGTTTCAGGGTAAGAAGAACC AGCTCCTAGTAAAAAGACTATTGTGACTGAAAAGCGCGTCAGCGCAAAGCCGACCGCACA AAACGCACAAGGAGTTACAG > Pseudomonas aeruginosa ACGCGGCCAGGGTCTTCTCCTGCGAGATCATGCGCGGCGCGCCGCGCATGCCGGCGCCGC TGCTGGAACGCCTCGACCCCAGG GCTACACTAGTTTAAC CGGAACGCCGCCAGTGGATCG GCCTGCCCCAGCTATTGCTC Expectation-Maximization (EM) • Randomly guess the locations of each motif instance • Repeat until convergence – Calculate a new motif model from the motif instances – Calculate new locations for the motif instances from the motif model 7

  8. EM - Randomly guess the locations of each motif instance > Escherichia coli TTGATTCCCTGAATGCCCGCTTAGTGTAACACTACTGTAACCGGCATTTTCTGCTTTTCC TGCCGATATTTTTTCTTATCTACCTCACAAAGGTTAGCAATAACTGCTGGGAA AATTCCG AGTTAGTCG TTATATTCTAT > Haemophilus influenzae A TCTAACGGTACGGATT CTCCAAAGGCCTATGGAATCTTGTAGAATATGAAACGTTCTAA TAAATCATAAAGTTGGAGCAAACGCTCGGCATAAGTAGTAAGTGCCGTGCCTCCGCCATT AGTTACACTAGTGGGACACC > Vibrio cholerae ATTTGTGGCGGTTTTCAAATGCTTGGAGAATGGGTACATGATCCGCTTGGCATTGAAGGT GAGGCTGGCAGCAGCGAAGGTCTGGGGCTGTTTGAACGTTACACGAGTGTAACCGCCGAA CC ATGTTGACACGAATTC TG > Salmonella typhi GGTCGGCTTAGACTAGTGTGACCAAAAAGCTTTTGCTGAAGTTTCAGGGTAAGAAGAACC AGCTCCTAGTAAAAAGACTAT TGTGACTGAAAAGCGC GTCAGCGCAAAGCCGACCGCACA AAACGCACAAGGAGTTACAG > Pseudomonas aeruginosa ACGCGGCCAGGGTCTTCTCCTGCGAGATCATGCGCGGCGCGCCGCGCATG CCGGCGCCGC TGCTGG AACGCCTCGACCCCAGGGCTACACTAGTTTAACCGGAACGCCGCCAGTGGATCG GCCTGCCCCAGCTATTGCTC A .40 .20 0.0 .20 .40 0.0 .20 .20 .40 .40 .20 .60 .20 .20 0.0 0.0 C .20 .40 0.0 0.0 .40 .60 .20 .40 0.0 .40 .20 0.0 .20 0.0 .20 .40 0.0 .20 .40 .40 0.0 .40 .40 .40 .40 0.0 .20 .40 .60 .20 .40 .40 G T .40 .20 .60 .40 .20 0.0 .20 0.0 .20 .20 .40 0.0 0.0 .60 .40 .20 > Escherichia coli TTGATTCCCTGAATGCCCGCTTAGTGTAACACTACTGTAACCGGCATTTTCTGCTTTTCC TGCCGATATTTTTTCTTATCTACCTCACAAAGGTTAGCAATAACTGCTGGGAA AATTCCG AGTTAGTCG TTATATTCTAT > Haemophilus influenzae A TCTAACGGTACGGATT CTCCAAAGGCCTATGGAATCTTGTAGAATATGAAACGTTCTAA TAAATCATAAAGTTGGAGCAAACGCTCGGCATAAGTAGTAAGTGCCGTGCCTCCGCCATT AGTTACACTAGTGGGACACC > Vibrio cholerae ATTTGTGGCGGTTTTCAAATGCTTGGAGAATGGGTACATGATCCGCTTGGCATTGAAGGT GAGGCTGGCAGCAGCGAAGGTCTGGGGCTGTTTGAACGTTACACGAGTGTAACCGCCGAA CC ATGTTGACACGAATTC TG > Salmonella typhi GGTCGGCTTAGACTAGTGTGACCAAAAAGCTTTTGCTGAAGTTTCAGGGTAAGAAGAACC AGCTCCTAGTAAAAAGACTAT TGTGACTGAAAAGCGC GTCAGCGCAAAGCCGACCGCACA AAACGCACAAGGAGTTACAG > Pseudomonas aeruginosa ACGCGGCCAGGGTCTTCTCCTGCGAGATCATGCGCGGCGCGCCGCGCATG CCGGCGCCGC TGCTGG AACGCCTCGACCCCAGGGCTACACTAGTTTAACCGGAACGCCGCCAGTGGATCG GCCTGCCCCAGCTATTGCTC 8

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