Control of Gene Expression

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

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

– 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


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)



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


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



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



Gene Structure and Gene Regulation in Eukaryotes

Drosophila Genome Organization


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



• 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



• 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



• 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)


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


Duplicate Genes Resolved


Gene Merge


Gene Split


Gene Merger/Split


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)


Overlapping Genes (UTR)


Alternative Splicing/Independent Proteins


Dicistronic Transcript


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


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

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#1-??, #2-DRE


#3-TATA, #4-INR


#9-DPE,#10-vDPE


Location of Promoter Elements

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Control of Gene Expression