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Control of Gene Expression

Control of Gene Expression. Prokaryotes and Operons. Regulated Gene Expression: an advantage. Lactose metabolism disaccharide) - made of glucose & galactose its oxidation provides cell with intermediates & energy lactose absent, then no B-galactosidase

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Control of Gene Expression

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  1. Control of Gene Expression Prokaryotes and Operons Karp/CELL & MOLECULAR BIOLOGY 3E

  2. Regulated Gene Expression: an advantage • Lactose metabolism • disaccharide) - made of glucose & galactose • its oxidation provides cell with intermediates & energy • lactose absent, then no B-galactosidase • lactose present, enzyme levels rise ~1000-fold • Tryptophan - essential amino acid; if not there, must be produced by bacterium at energy cost; needed for protein synthesis • if absent, cells make tryptophan • if present, genes repressed within minutes Karp/CELL & MOLECULAR BIOLOGY 3E

  3. Figure 12.24 Karp/CELL & MOLECULAR BIOLOGY 3E

  4. Bacterial operon - Jacob & Monod (Pasteur Inst., 1961) • Components of operon (single mRNA) • Structural genes - code for operon enzymes • Promoter • Operator - between promoter & genes • Repressor – binds to operator • Regulatory gene - codes for repressor protein • Repressor is key • it binds to operator, shielding promoter • Repressor regulated allosterically • presence or absence of inducer (lactose or tryptophan) Karp/CELL & MOLECULAR BIOLOGY 3E

  5. Figure 12.25 Karp/CELL & MOLECULAR BIOLOGY 3E

  6. The lac operon - inducible operon • What are the structural genes in the lac operon? • z gene - encodes B-galactosidase • y gene - encodes galactoside permease; promotes lactose entry into cell • a gene - encodes thiogalactoside acetyltransferase; role is unclear • Inducible operon • If lactose present, binds repressor, changing its shape • Repressor binds promoter only in absence of inducer Karp/CELL & MOLECULAR BIOLOGY 3E

  7. The lac operon - inducible operon • Positive control by cyclic AMP • Glucose inhibits lac expression • cAMP inversely related to amount of glucose in medium • cAMP activates lac • cAMP binds to cAMP receptor protein (CRP) • CRP binds DNA only if cAMP bound • CRP-cAMP complex allows RNA polymerase to transcribe • cAMP-CRP complex is necessary for lac operon transcription Karp/CELL & MOLECULAR BIOLOGY 3E

  8. Figure 12.27 Karp/CELL & MOLECULAR BIOLOGY 3E

  9. The trp operon - a repressible operon • repressor is unable to bind to operator DNA by itself • Repressor active only if bound by corepressor (tryptophan) • Without tryptophan, operon is expressed • Trp operon also regulated by attenuation: conditional termination Karp/CELL & MOLECULAR BIOLOGY 3E

  10. Figure 12.26 Karp/CELL & MOLECULAR BIOLOGY 3E

  11. Gene Structure and Gene Regulation in Eukaryotes Drosophila Genome Organization Karp/CELL & MOLECULAR BIOLOGY 3E

  12. Annotation 3 for Flys • cDNA’s now identified for 78% of genes • helpful for defining introns, start sites, etc. • Compared with release 2 • 85% of transcripts changed • 45% of proteins changed • added transposons and RNA genes • found many unusual genes Karp/CELL & MOLECULAR BIOLOGY 3E

  13. Annotation 3 for Flys • transcripts predicted using • Genie, Genescan gene prediction softwares • Similarity to proteins using BLASTX • Similarity to translated cDNA’s using TBLASTX • DNA alignments to cDNA’s • 116.8Mb euchromatin; 20.7 Mb heterochromatin • Found more exons and introns • Found more 5’ and 3’ UTR’s • 20% of genes are alternatively spliced Karp/CELL & MOLECULAR BIOLOGY 3E

  14. Annotation 3 for Flys • Transposons (1,572) • 682 LTR • 486 LINE • 372 TIR • 32 FB (foldback elements) • 28 snRNA’s (for splicing) • 28 snoRNA’s (7SLRNA, RNAse P RNA) • 27 new longer RNA genes from cDNA Karp/CELL & MOLECULAR BIOLOGY 3E

  15. Annotation 3 for Flys • 17 pseudogenes (15 simple recombination, 1 is processed, 1 is very diverged) • 802 new protein coding genes • Resolved some repeated genes (Trypsin) • 345 genes from release 2 rejected (<50 aa’s, predicted only) Karp/CELL & MOLECULAR BIOLOGY 3E

  16. New gene models • Gene Duplicates (Fig1) • Gene Merges (Fig 3) • Gene Splits (Fig 4) • Gene Split/Merges (Fig 5) • Nested genes (7.5% of all genes are in introns) • 26 “interleaved” (alternating introns, exons) • 431 transposons in introns Karp/CELL & MOLECULAR BIOLOGY 3E

  17. Duplicate Genes Resolved Karp/CELL & MOLECULAR BIOLOGY 3E

  18. Gene Merge Karp/CELL & MOLECULAR BIOLOGY 3E

  19. Gene Split Karp/CELL & MOLECULAR BIOLOGY 3E

  20. Gene Merger/Split Karp/CELL & MOLECULAR BIOLOGY 3E

  21. New gene models • Overlapping genes • 15% on opposite strand (mostly UTR: antisense regulation?) • 60 cases overlap on same strand (Fig 6) • Alternatively spliced • 21 lola transcripts and 29 mod(mdg4) transcripts: • both are RNA pol II factors – pleiotropy • 2 genes have non-overlapping protein products • 31 discistronic (IRS or reinitiation) Karp/CELL & MOLECULAR BIOLOGY 3E

  22. Overlapping Genes (UTR) Karp/CELL & MOLECULAR BIOLOGY 3E

  23. Alternative Splicing/Independent Proteins Karp/CELL & MOLECULAR BIOLOGY 3E

  24. Dicistronic Transcript Karp/CELL & MOLECULAR BIOLOGY 3E

  25. Core Promoters in Drosophila • Cap-trapped cDNA 5’ ends • TATA, INITIATOR, DPE, vDPE. DRE • Used to retrain MacPromoter • 1,941 TSS’s (11 base window) • Covers 14% of all genes • About 550 promotors already well described • Only 18% of new promoters matched old promoters • Only 30 seemed to have different TSS Karp/CELL & MOLECULAR BIOLOGY 3E

  26. Core Promoter Elements of Flys Table 2 ------------------------------------------------------------------------ The ten most significant motifs in the core promoter sequences from -60 to +40, as identified by the MEME algorithm ------------------------------------------------------------------------ Motif Pictogram Bits Consensus Number E value ------------------------------------------------------------------------ 1 [Image] 15.2 YGGTCACACTR 311 5.1e-415 2 DRE [Image] 13.3 WATCGATW 277 1.7e-183 3 TATA [Image] 13.2 STATAWAAR 251 2.1e-138 4 INR [Image] 11.6 TCAGTYKNNNTYNR 369 3.4e-117 5 [Image] 15.2 AWCAGCTGWT 125 2.9e-93 6 [Image] 15.1 KTYRGTATWTTT 107 1.9e-62 7 [Image] 12.7 KNNCAKCNCTRNY 197 1.9e-63 8 [Image] 14.7 MKSYGGCARCGSYSS 82 5.1e-29 9 DPE [Image] 15.4 CRWMGCGWKCGGTTS 56 1.9e-12 10 vDPE [Image] 15.3 CSARCSSAACGS 40 8.3e-9 ------------------------------------------------------------------------ Karp/CELL & MOLECULAR BIOLOGY 3E

  27. #1-??, #2-DRE Karp/CELL & MOLECULAR BIOLOGY 3E

  28. #3-TATA, #4-INR Karp/CELL & MOLECULAR BIOLOGY 3E

  29. #9-DPE,#10-vDPE Karp/CELL & MOLECULAR BIOLOGY 3E

  30. Location of Promoter Elements Karp/CELL & MOLECULAR BIOLOGY 3E

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