30
Copyright, ©, 2002, John Wiley & Sons, Inc., Karp/CELL & MOLECULAR BIOLOGY 3E Control of Gene Expression Prokaryotes and Operons

Control of Gene Expression

  • Upload
    yvonne

  • View
    54

  • Download
    0

Embed Size (px)

DESCRIPTION

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 - PowerPoint PPT Presentation

Citation preview

Page 1: Control of Gene Expression

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

Control of Gene Expression

Prokaryotes and Operons

Page 2: Control of Gene Expression

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

Page 3: Control of Gene Expression

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

Page 4: Control of Gene Expression

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) 

Page 5: Control of Gene Expression

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

Page 6: Control of Gene Expression

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

Page 7: Control of Gene Expression

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

Page 8: Control of Gene Expression

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

Page 9: Control of Gene Expression

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

Page 10: Control of Gene Expression

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

Page 11: Control of Gene Expression

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

Gene Structure and Gene Regulation in Eukaryotes

Drosophila Genome Organization

Page 12: Control of Gene Expression

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

Page 13: Control of Gene Expression

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

Page 14: Control of Gene Expression

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

Page 15: Control of Gene Expression

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)

Page 16: Control of Gene Expression

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

Page 17: Control of Gene Expression

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

Duplicate Genes Resolved

Page 18: Control of Gene Expression

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

Gene Merge

Page 19: Control of Gene Expression

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

Gene Split

Page 20: Control of Gene Expression

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

Gene Merger/Split

Page 21: Control of Gene Expression

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)

Page 22: Control of Gene Expression

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

Overlapping Genes (UTR)

Page 23: Control of Gene Expression

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

Alternative Splicing/Independent Proteins

Page 24: Control of Gene Expression

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

Dicistronic Transcript

Page 25: Control of Gene Expression

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

Page 26: Control of Gene Expression

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

------------------------------------------------------------------------

Page 27: Control of Gene Expression

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

#1-??, #2-DRE

Page 28: Control of Gene Expression

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

#3-TATA, #4-INR

Page 29: Control of Gene Expression

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

#9-DPE,#10-vDPE

Page 30: Control of Gene Expression

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

Location of Promoter Elements