Mutations Affecting Regulation of the lac Operon

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Mutations Affecting Regulation of the lac Operon

• Mutations leading to constitutive expression of
  lac operon genes
• lacZ, lacY, lacA transcribed even in absence of
  lactose
• Mutations leading to an uninducible lac operon
• lacZ, lacY, lacA not transcribed even in presence
  of lactose

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Consitutive Mutations

• lacI-
• No repressor
• lacI-d
• Defective repressor cant bind operator
• Oc
• Mutant operator cant bind repressor

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Uninducible Mutations

• lacIs
• Defective repressor cant bind inducer

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Complementation of Mutations in Bacteria

• Bacteria are haploid
• There is no second allele to compensate for
  mutation
• Bacteria can be made into partial diploids (aka
  merodiploid) with plasmid containing gene(s) of
  interest

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Jacob and Monod

• Identified bacteria with mutations affecting
  regulation of lac operon
• Created partial diploids by introducing plasmid
  containing wild-type lac operon genes (lacI,
  lacZ, lacY, lacA)
• Assayed for b-galactosidase as a measure of gene
  transcription from lac operon promoter in
  presence or absence of lactose

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lacI- is recessive in merodiploids

• Wt allele produces repressor which can act in
  trans on mutant allele to repress transcription

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Oc is cis-dominant in merodiploid

• Wt allele is repressible, but mutant allele is
  consitutively active
• Mutation in operator site works in cis and is
  dominant over wt allele

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lacI-d is dominant-negative in merodiploid

• Mutant and wt repressors form heterotetramers,
  which can not bind operator on either allele
• Transcription is always on, even in absence of
  inducer
• Mutation is dominant over wt, negatively
  affecting function of wt

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lacIs is cis- and trans-dominant in merodiploid

• Mutant and wt repressors form heterotetramers
  which can bind operator but can not bind inducer
• Repressor remains bound to operator site on both
  alleles even in presence of inducer (allolactose)
• Mutation affects both the cis and the trans allele

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The trp Operon
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The trp Operon

• 5 genes required for biosynthesis of aa
  tryptophan (an anabolic process)
• Genes transcribed as single long polycistronic
  message proteins translated independently
• Genes expressed when trp levels are low

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When trp levels are high, trp represses its own
synthesis..
Trp binds to an aporepressor dimer Trp-bound
dimer now acts as a repressor Repressor dimer
binds operator site Operator site lies within
(overlaps) the promoter site Trp is considered
a corepressor
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When trp levels are low
Trp dissociates from repressor Aporepressor can
not bind operator in absence of trp Operon is
derepressed RNA pol binds promoter Transcription
of trp operon genes
No requirement for a secondary activator
protein Derepression is sufficient to promote
transcription from the trpP promoter
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Repression of the trp operon is weak Even when
trp is available, transcription occurs at a
fairly high level An additional mechanism is
required to prematurely terminate transcription
that is initiated in the presence of trp
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The trpL (leader) and trpa (attenuator) region
transcribed first Transcription initiated in the
presence of trp is prematurely terminated at the
attenuator
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Nascent RNA transcribed from attenuator will
form intrinsic terminator The hairpin will cause
the RNA pol to stall Weak base-pairing between
dA and rU is disrupted Release of unfinished
RNA Termination of transcription
Intrinsic terminator present in attenuator
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To terminate or not to terminate? How can there
be any transcription of the trp biosynthetic
genes if there is an intrinsic terminator
upstream? When trp levels are low, RNA pol does
not terminate, but reads through trp biosynthetic
genes
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Hairpin 1
polyU
Hairpin 2 Intrinsic Terminator
Hairpin 3
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If hairpins 1 and 2 are allowed to
form ATTENUATION (termination of
transcription) If hairpin 3 is allowed to
form TRANSCRIPTION What determines which of
the hairpins will form? TRYPTOPHAN
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The leader region (trpL) contains a small ORF
coding for a 14 aa peptide Because of coupled
transcription-translation in prokaryotes, this
peptide is translated as soon as the mRNA for it
is transcribed.
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very rare!
The transcribed leader sequence serves as a trp
sensor If trp is low, the ribosome (and
translation) will stall
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Trp codons
In absence of Trp, ribosome stalls over region
required for formation of hairpin 1 Hairpin 1
can not form As RNA pol continues to
transcribe, Hairpin 3 forms Hairpin 2 can not
form No attenuation
Hairpin 1
Hairpin 2
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Hairpin 2 Instrinsic Terminator
Hairpin 1
Hairpin 3
Mechanism depends on coupled transcription-transla
tion and the rate at which it occurs
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Identification of Binding Sites in DNA for
DNA-Binding Proteins

• DNA Footprinting (also called nuclease protection
  assay)
• Label end of DNA
• Incubate with DNA-binding protein
• Cut protein-complexed DNA with nucleases
• Identify regions of DNA protected by the
  protein
• Gel Mobility Shift Assay
• Label end of DNA
• Incubate with DNA-binding protein
• Run complex out on gel
• DNA with bound protein will migrate more slowly

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DNA Footprinting
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Gel Mobility Shift Assay