A1261980008oXnNm

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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
lacZ - ?-galactosidase breaks lactose into
galactose and glucose lacY - permease is
required to transport lactose into the cell lacA
- transacetylase, not required but can
assay Lac- mutants were isolated in Z and Y,
prevent utilization
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 diffusible factor
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Analysis of lacOc mutants
lacO is cis acting - a non-diffusible element
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Analysis of lacP mutants
lacP is cis acting - a non-diffusible element
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Regulatory models for the lac operon
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LacI binds DNA as a tetramer to repress
transcription
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Genetic analysis of the LacI binding sites
Why were these sites not identified in the
screens done by Jacob and Monod?
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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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Nonsense suppressors
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Polar mutations affect genes downstream
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Mutational analysis of Repression and Activation
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Effect of inducers on regulation
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Crossfeeding analysis of Trp mutants allows to
analyze the biochemistry of Trp biosynthesis
pathway
TrpE
TrpD
TrpB
precursor
Trp
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(No Transcript)
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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
What would the phenotype be of a deletion from
120-130?
What would the phenotype be of the Trp codon
deletions?
Can this form of regulation happen in eukaryotes?