Control of Gene Expression Prokaryotes and Operons Copyright

Control of Gene Expression Prokaryotes and Operons Copyright, ©, 2002, John Wiley & Sons, Inc. , Karp/CELL & MOLECULAR BIOLOGY 3 E

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 3 E

Copyright, ©, 2002, John Wiley & Sons, Inc. , Karp/CELL & MOLECULAR BIOLOGY 3 E Figure 12. 24

Bacterial operon - Jacob & Monod (Pasteur Inst. , 1961) • Components of operon (single m. RNA) – 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 3 E

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

The lac operon - inducible operon • Positive control by cyclic AMP – Glucose inhibits lac expression – c. AMP inversely related to amount of glucose in medium – c. AMP activates lac – c. AMP binds to c. AMP receptor protein (CRP) – CRP binds DNA only if c. AMP bound – CRP-c. AMP complex allows RNA polymerase to transcribe – c. AMP-CRP complex is necessary for lac operon transcription Copyright, ©, 2002, John Wiley & Sons, Inc. , Karp/CELL & MOLECULAR BIOLOGY 3 E

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

Copyright, ©, 2002, John Wiley & Sons, Inc. , Karp/CELL & MOLECULAR BIOLOGY 3 E Figure 12. 26

Gene Structure and Gene Regulation in Eukaryotes Drosophila Genome Organization Copyright, ©, 2002, John Wiley & Sons, Inc. , Karp/CELL & MOLECULAR BIOLOGY 3 E

Annotation 3 for Flys • c. DNA’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 3 E

Annotation 3 for Flys • transcripts predicted using – – Genie, Genescan gene prediction softwares Similarity to proteins using BLASTX Similarity to translated c. DNA’s using TBLASTX DNA alignments to c. DNA’s • 116. 8 Mb 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 3 E

Annotation 3 for Flys • • Transposons (1, 572) 682 LTR 486 LINE 372 TIR 32 FB (foldback elements) 28 sn. RNA’s (for splicing) 28 sno. RNA’s (7 SLRNA, RNAse P RNA) 27 new longer RNA genes from c. DNA Copyright, ©, 2002, John Wiley & Sons, Inc. , Karp/CELL & MOLECULAR BIOLOGY 3 E

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 3 E

New gene models • • • Gene Duplicates (Fig 1) 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 3 E

Duplicate Genes Resolved Copyright, ©, 2002, John Wiley & Sons, Inc. , Karp/CELL & MOLECULAR BIOLOGY 3 E

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

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

Gene Merger/Split Copyright, ©, 2002, John Wiley & Sons, Inc. , Karp/CELL & MOLECULAR BIOLOGY 3 E

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(mdg 4) 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 3 E

Overlapping Genes (UTR) Copyright, ©, 2002, John Wiley & Sons, Inc. , Karp/CELL & MOLECULAR BIOLOGY 3 E

Alternative Splicing/Independent Proteins Copyright, ©, 2002, John Wiley & Sons, Inc. , Karp/CELL & MOLECULAR BIOLOGY 3 E

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

Core Promoters in Drosophila • • • Cap-trapped c. DNA 5’ ends TATA, INITIATOR, DPE, v. DPE. DRE Used to retrain Mac. Promoter 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 3 E

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. 1 e-415 2 DRE [Image] 13. 3 WATCGATW 277 1. 7 e-183 3 TATA [Image] 13. 2 STATAWAAR 251 2. 1 e-138 4 INR [Image] 11. 6 TCAGTYKNNNTYNR 369 3. 4 e-117 5 [Image] 15. 2 AWCAGCTGWT 125 2. 9 e-93 6 [Image] 15. 1 KTYRGTATWTTT 107 1. 9 e-62 7 [Image] 12. 7 KNNCAKCNCTRNY 197 1. 9 e-63 8 [Image] 14. 7 MKSYGGCARCGSYSS 82 5. 1 e-29 9 DPE [Image] 15. 4 CRWMGCGWKCGGTTS 56 1. 9 e-12 10 v. DPE [Image] 15. 3 CSARCSSAACGS 40 8. 3 e-9 ------------------------------------ Copyright, ©, 2002, John Wiley & Sons, Inc. , Karp/CELL & MOLECULAR BIOLOGY 3 E

#1 -? ? , #2 -DRE Copyright, ©, 2002, John Wiley & Sons, Inc. , Karp/CELL & MOLECULAR BIOLOGY 3 E

#3 -TATA, #4 -INR Copyright, ©, 2002, John Wiley & Sons, Inc. , Karp/CELL & MOLECULAR BIOLOGY 3 E

#9 -DPE, #10 -v. DPE Copyright, ©, 2002, John Wiley & Sons, Inc. , Karp/CELL & MOLECULAR BIOLOGY 3 E

Location of Promoter Elements Copyright, ©, 2002, John Wiley & Sons, Inc. , Karp/CELL & MOLECULAR BIOLOGY 3 E