Copyright, ©, 2002, John Wiley & Sons, Inc.,Karp/CELL & MOLECULAR BIOLOGY 3E 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 Figure 12.24

Copyright, ©, 2002, John Wiley & Sons, Inc.,Karp/CELL & MOLECULAR BIOLOGY 3E 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 Figure 12.25

Copyright, ©, 2002, John Wiley & Sons, Inc.,Karp/CELL & MOLECULAR BIOLOGY 3E 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 Figure 12.27

Copyright, ©, 2002, John Wiley & Sons, Inc.,Karp/CELL & MOLECULAR BIOLOGY 3E 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 Figure 12.26

Copyright, ©, 2002, John Wiley & Sons, Inc.,Karp/CELL & MOLECULAR BIOLOGY 3E 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 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 [Image] 15.2 YGGTCACACTR e DRE [Image] 13.3 WATCGATW e TATA [Image] 13.2 STATAWAAR e INR [Image] 11.6 TCAGTYKNNNTYNR e [Image] 15.2 AWCAGCTGWT e-93 6 [Image] 15.1 KTYRGTATWTTT e-62 7 [Image] 12.7 KNNCAKCNCTRNY e-63 8 [Image] 14.7 MKSYGGCARCGSYSS e-29 9 DPE [Image] 15.4 CRWMGCGWKCGGTTS e vDPE [Image] 15.3 CSARCSSAACGS e

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