P1258791345xvGtP

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The trp operon
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Reading MVA pp. 760-767 and 1011-1018 Problem
Ch 26 9 Alberts pp. 395-399
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MVA Fig.21.1
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MVA Fig.21.12
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MVA Fig.21.13
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MVA Fig.21.14
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MVA Fig.21.15
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The chromosomal order of genes in the trp operon
of E. coli and the sequence of reactions
catalyzed by the enzyme products of the trp
structural genes. The products of genes trpD and
trpE form a complex that catalyzes specific
steps, as do the products of genes trpB and trpA.
Tryptophan synthetase is a tetrameric enzyme
formed by the products of trpB and trpA. It
catalyzes a two-step process leading to the
formation of tryptophan. (PRPP, phosphoribosyl
pyrophosphate CDRP, 1-(o-carboxyphenylamino)-1-de
oxyribulose 5-phosphate.) (After S. Tanemura and
R. H. Bauerle, Genetics 95, 1980, 545.)
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MVA Fig. 26.33
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Alberts Fig. 7-34
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Originally, regulation of the trp operon was
thought to occur solely through the
repressor-operator system until deletion mutants
located downstream of trpO were identified.
These mutants displayed increased expression of
the operon by six-fold which indicated the
presence of an additional transcriptional control
element.
Why is repression not the only mode of regulation?
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Evidence 1. Trp-tRNA synthetase mutants had
regulatory anomalies. 2. Addition of trp to
trp-starved cells not only shut down initiation
of transcription but also inhibited transcription
already in progress on the initial segment of
the operon. 3. Mutants lacking a functional
repressor could still respond to trp starvation
by increasing transcription of trp mRNA. 4 .
Deletion mutants in which both of the deletion
termini were within the transcribed region of
the operon had an unexpected six-fold increase
in expression of the remaining genes in the
operon. Obviously, repressor binding was
unaffected.
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5. Within the population of mRNAs produced in
vivo from the 5' end of the trp operon, RNAs
corresponding to the first 140 bp (the leader
sequence) of the operon were several times more
abundant than those from more distal regions,
therefore a transcription termination site was
located before the structural genes.
6. Starving bacteria of trp reduced termination
at this site (the trp attenuator).
7. Mutations altering trp-tRNA synthetase,
tRNAtrp or a tRNA trp modifying enzyme were
found to decrease transcription termination at
the trp attenuator. What does this suggest
about the mode of attenuation?
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8. Ribosome binding experiments with the 140
base transcript demonstrated that ribosomes
protect a 20 base segment from nuclease attack.
A potential AUG start codon is located in the
center of this region. 9. A 14 residue peptide
(the leader peptide) could be synthesized from
this start codon and contained tandem trp
residues near its C-terminus. 10. The trp
leader ribosome binding site was shown to be an
efficient site for the initiation of translation
by fusing the leader to a structural gene and
demonstrating synthesis of the fused
polypeptide.
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11. Two classes of termination defective leader
mutants have been isolated. One type terminates
at less than normal frequency and has bp changes
in the 34 bp region. In vivo, these mutants
have a 2-4 fold increase in operon expression.
12. The second class of mutants have increased
termination of the attenuator. These prevent the
relief from termination that is associated with
trp starvation. One of these mutants has
an altered start codon for the leader peptide.
Another has a G to A conversion at position 75,
which would prevent 23 pairing and cause
formation of a 34 termination structure.
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Genetic analysis indicated that the new control
element was located in trpL, a 162 nt region
30-60 nt upstream from trpE. When trp is
scarce, the entire 6720 nt polycistronic trp,
including trpL, is synthesized. As the trp
concentration increases, the rate of trp
transcription decreases as a result of the trp
r epressor-corepressor's greater abundance.
With increasing trp, the mRNA synthesized
consists more and more of a 140 nt segment
corresponding to trpL sequences only. The
availability of trp results in the premature
termination of transcription of the operon.
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MVA Fig. 26.35
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MVA Fig. 26.36
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MVA Fig.21.17
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MVA Fig. 26.37
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MVA Fig. 26.39
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MVA Fig. 26.4