Transcript Document

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
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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
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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
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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
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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
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Karp/CELL & MOLECULAR BIOLOGY 3E
Gene Merge
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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
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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)
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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
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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
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Karp/CELL & MOLECULAR BIOLOGY 3E
#9-DPE,#10-vDPE
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Karp/CELL & MOLECULAR BIOLOGY 3E
Location of Promoter Elements
Copyright, ©, 2002, John Wiley & Sons, Inc.,
Karp/CELL & MOLECULAR BIOLOGY 3E