Prokaryotic Genetics III Gene regulation

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Prokaryotic Genetics III - Gene regulation
1. Lac System Jacob and Monod, 1950's, biochemists
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Non-metabolic inducer
Non-inducer substrates
X-gal
ONPG
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Ampicillin enrichment of lac- cells
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Lac- mutants - genes mapped close together
Lac- mutants were isolated in Z and Y, prevent
utilization lacZ - ?-galactosidase breaks
lactose into galactose and glucose lacY -
permease is required to transport lactose into
the cell
One mutant allowed expression in the absence of
inducer constitutive mutant, lacI First example
of a mutant that affects production not activity
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PaJaMo Experiment - Pardee, Jacob Monond
Hfr I Z T6sSs x F- I-Z-T6rSr
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Analysis of lacI mutants
LacI is trans acting - a diffusable factor
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Analysis of lacOc mutants
lacO is cis acting - a non-diffusable element
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Analysis of lacP mutants
lacP is cis acting - a non-diffusable element
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Regulatory models for the lac operon
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LacI binds DNA as a tetramer to repress
transcription
Why did Jacob Monod not find O2 and O3?
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Genetic analysis of the LacI binding sites
Blue
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Distinction between factors (proteins) and
elements (DNA sites)
i) Regulatory factors act in trans
ii) Regulatory elements act in cis
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Mutational analysis of Repression and Activation
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Effect of inducers on regulation
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Cross feeding analysis of Trp mutants allows to
analyze the biochemistry of Trp biosynthesis
pathway
TrpE
TrpD
TrpB
precursor
Trp
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TrpE
TrpD
TrpC1
TrpC2
TrpA
TrpB
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Attenuation of trp operator expression
attenuator
Deletions in the attenuator increase basal
synthesis of Trp enzymes
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Sequences Coding for Short Peptides are Often
Found in Leaders of Transcripts from Biosynthesis
Operons
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trp Operon expression in response to Trp levels
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Sequences in the trp operon leader region can
form different stem-loop structures
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Stalled ribosomes block the formation of the
transcription terminating stem-loop structure
Trp
-Trp
Can this form of regulation happen in eukaryotes?
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Life cycle of a Temperate Bacteriophage
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Mating by lysogen induces lytic growth in
recipient cell
Similar to the PaJaMa Experiment
Hfr
F-
No repressor of the prophage in the recipient
cell causes lytic growth
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Screen for clear plaques
How did they define the complementation groups?
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Different complementation groups and phenotypes
of the "clear" mutants
Recessive trans
Dominant - cis
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Infection of WT phage into a lysogen
Uninfected
Lysogen
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Infection of cI phage into a lysogen
Uninfected
Lysogen
Recessive trans
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Infection of cI phage into a lysogen
Uninfected
Lysogen
Recessive trans
Dominant - cis
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? is linear in the virion but circularizes after
infection
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? phage genome DNA replication
(2) Recombination (10) Head (10), tail
(12) lysis (3)
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Differential gene expression in the ? life cycle
Integrate into genome
cI
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Regulation of early gene expression
N (?) and Nus (bacteria) function as
anti-terminators
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Regulation of late lytic gene expression
26 Kb, takes 10'
Shuts off genes required for lysogeny
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Regulation of late lysogenic and prophage gene
expression
cI shuts of all other phage genes in Prophage
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Lytic vs lysogen decision
To establish lysogen
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Induction of prophage through SOS response
cI
Does not bind DNA
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Integration and excision of ? phage
Int/Xis ratio determines direction
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To establish lysogeny Int gt Xis
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To establish lytic growth Xis gt Int
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Lytic induction from a lysogen Xis Int
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Construction of a ? phage library
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Construction of a Cosmid library