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Control of Gene Expression

This overview explores the regulation of gene expression in prokaryotes, focusing on the lac and trp operons as key examples. It discusses how lactose and tryptophan influence enzyme production, detailing the roles of structural genes, promoters, operators, and repressors in operon function. The mechanisms of positive and negative control are examined, highlighting the cyclic AMP relationship with glucose in lactase expression. By understanding operon models, we can appreciate the intricate regulatory processes that enable bacterial adaptability to environmental changes.

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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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