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Control of Gene Expression Prokaryotes and Operons Copyright, ©, 2002, John Wiley & Sons, Inc., Karp/CELL & MOLECULAR BIOLOGY 3E 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 Copyright, ©, 2002, John Wiley & Sons, Inc., Karp/CELL & MOLECULAR BIOLOGY 3E Copyright, ©, 2002, John Wiley & Sons, Inc., Karp/CELL & MOLECULAR BIOLOGY 3E Figure 12.24 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) Copyright, ©, 2002, John Wiley & Sons, Inc., Karp/CELL & MOLECULAR BIOLOGY 3E Copyright, ©, 2002, John Wiley & Sons, Inc., Karp/CELL & MOLECULAR BIOLOGY 3E Figure 12.25 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 Copyright, ©, 2002, John Wiley & Sons, Inc., Karp/CELL & MOLECULAR BIOLOGY 3E 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 Copyright, ©, 2002, John Wiley & Sons, Inc., Karp/CELL & MOLECULAR BIOLOGY 3E Copyright, ©, 2002, John Wiley & Sons, Inc., Karp/CELL & MOLECULAR BIOLOGY 3E Figure 12.27 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 Copyright, ©, 2002, John Wiley & Sons, Inc., Karp/CELL & MOLECULAR BIOLOGY 3E Copyright, ©, 2002, John Wiley & Sons, Inc., Karp/CELL & MOLECULAR BIOLOGY 3E Figure 12.26 Gene Structure and Gene Regulation in Eukaryotes Drosophila Genome Organization Copyright, ©, 2002, John Wiley & Sons, Inc., Karp/CELL & MOLECULAR BIOLOGY 3E 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 Copyright, ©, 2002, John Wiley & Sons, Inc., Karp/CELL & MOLECULAR BIOLOGY 3E 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 Copyright, ©, 2002, John Wiley & Sons, Inc., Karp/CELL & MOLECULAR BIOLOGY 3E 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 Copyright, ©, 2002, John Wiley & Sons, Inc., Karp/CELL & MOLECULAR BIOLOGY 3E 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) Copyright, ©, 2002, John Wiley & Sons, Inc., Karp/CELL & MOLECULAR BIOLOGY 3E 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 Copyright, ©, 2002, John Wiley & Sons, Inc., Karp/CELL & MOLECULAR BIOLOGY 3E Duplicate Genes Resolved Copyright, ©, 2002, John Wiley & Sons, Inc., Karp/CELL & MOLECULAR BIOLOGY 3E Gene Merge Copyright, ©, 2002, John Wiley & Sons, Inc., Karp/CELL & MOLECULAR BIOLOGY 3E Gene Split Copyright, ©, 2002, John Wiley & Sons, Inc., Karp/CELL & MOLECULAR BIOLOGY 3E Gene Merger/Split Copyright, ©, 2002, John Wiley & Sons, Inc., Karp/CELL & MOLECULAR BIOLOGY 3E 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) Copyright, ©, 2002, John Wiley & Sons, Inc., Karp/CELL & MOLECULAR BIOLOGY 3E Overlapping Genes (UTR) Copyright, ©, 2002, John Wiley & Sons, Inc., Karp/CELL & MOLECULAR BIOLOGY 3E Alternative Splicing/Independent Proteins Copyright, ©, 2002, John Wiley & Sons, Inc., Karp/CELL & MOLECULAR BIOLOGY 3E Dicistronic Transcript Copyright, ©, 2002, John Wiley & Sons, Inc., Karp/CELL & MOLECULAR BIOLOGY 3E 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 Copyright, ©, 2002, John Wiley & Sons, Inc., Karp/CELL & MOLECULAR BIOLOGY 3E 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 ------------------------------------------------------------------------ Copyright, ©, 2002, John Wiley & Sons, Inc., Karp/CELL & MOLECULAR BIOLOGY 3E #1-??, #2-DRE Copyright, ©, 2002, John Wiley & Sons, Inc., Karp/CELL & MOLECULAR BIOLOGY 3E #3-TATA, #4-INR Copyright, ©, 2002, John Wiley & Sons, Inc., Karp/CELL & MOLECULAR BIOLOGY 3E #9-DPE,#10-vDPE Copyright, ©, 2002, John Wiley & Sons, Inc., Karp/CELL & MOLECULAR BIOLOGY 3E Location of Promoter Elements Copyright, ©, 2002, John Wiley & Sons, Inc., Karp/CELL & MOLECULAR BIOLOGY 3E