Sin t

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Prokaryotic promoters in the test tube and in
the environment
Pseudomonas putida
Víctor de Lorenzo Centro Nacional de
Biotecnología CSIC
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Enrichment cultures
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Strain (Plasmid) Pollutant P. putida mt-2
(pWW0) Toluene Pseudomonas sp, CF600
(pVI150) Phenol Burkholderia sp.
RP007 Phenantrene P. putida TMB Trimethyl
benzene P. putida NAH (NAH7) Naphthalene Acineto
bacter sp. ADP1 Aryl esters R. eutropha
(pJP4) 2,4 D Pseudomonas sp.
(pP51) Trichlorobenzene P. putida UCC22
(pTDN1) Aniline P. oleovorans
(pOCT) n-alkanes Burkholderia cepacia
LB400 Biphenyl, PCBs Pseudomonas sp.
VLB120 Styrene
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Evolution of biodegradative capabilities
1. Genes encoding catabolic enzymes 2.
Regulatory systems responding to substrates
3. Coupling transcription to Physiological
signals
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Stages in the regulation of gene expression
systems
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inducer
Expression of catabiolic genes
addition
C
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3
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Growth
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Evolutionary gravitation towards an optimal
regulation
Gain / capacity
Window of
activity
Specificity
Metabolic coupling
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And now a reminder on promoters and
gene expression in bacteria...
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Gene expression is a multi-step process
Transcription
mRNA stability
Translation
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Overview of transcription and translation
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Transcription is in itself a multi-step process
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Stem-loop structure of some terminators
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Bacterial genes are frequently clustered in
operons
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What is a promoter?
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Structure of rifampin
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Positive and negative regulation
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Regulation of transcription by two-component
regulatory systems
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Regulation of transcription by quorum-sensing
systems
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Structures of some autoinducers in quorum-sensing
systems
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s factors determine promoter specificity
Sigma 54
Sigma 70 family
A class in its own!
ECF Family Ec rpoE Ec rpoH Ec fecI
Sigma 70-like Ec rpoD Ec rpoS Ec rpoF
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Sigma 54 promoters
-12/-24 consensus sequence
Require an activator that binds far from 1
GG-10-GC
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The -12/-24 site
Small compared to -10/-35 GC rich (frequent
in Pseudomonas)
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Reporter lacZ gene fusions
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Monitoring promoter activity once at a time
Accumulation of beta-galactosidase
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The big leap forward from genetics to
genomics
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Computer-assisted search for genes and promoters
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Probing global gene expression
with Gene arrays
Condition A
Condition B
Differential gene Expression (DGE) reveals
signals and promoters
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Number of Regulators
450
400
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300
250
200
150
100
50
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Structural genes Enzymes Membrane proteins
Transcriptional regulators
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Regulation of translation
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Example of feedback inhibition
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Example of feedback inhibition