Single base-pair alterations in the Escherichia coli trp operon leader region that relieve transcription termination at the trp attenuator.
We have isolated a set of regulatory mutants defective in transcription termination at the attenuator in the leader region of the Escherichia coli tryptophan (trp) operon. In vivo the mutants have 2- to 4-fold increased levels of expression of the trp operon above the level of the trpR parental strain. These levels are increased an additional 1.5- to 2-fold when the mutants are starved of tryptophan. Transcription termination at the trp attenuator was analyzed in vitro with DNA restriction fragments containing the termination-relief mutations. Whereas the frequency of readthrough transcription beyond the termination site is 5% with the wild-type DNA template, it is 46-76% when mutant DNAs are used as templates. The base change in the leader region of each mutant was determined by RNA and/or DNA sequencing. All the changes were between base pairs +116 and +132, in the G-C-rich segment of the leader region. The RNA residues between +114 and +134 of the leader transcript can form a stable stem and loop structure [deltaG approximately equal to -20 kcal (-84 kJ)]. All of the termination-relief mutations destabilize this structure (deltaG approximately equal to -9.0 to -10.5 kcal). These results suggest that the efficiency of transcription termination may be dependent on the integrity of the secondary structure of the above segment of the transcript of the leader region.
D001483:Base Sequence; D004926:Escherichia coli / Q000235:genetics*; D009154:Mutation; D009876:Operon; D014158:Transcription, Genetic; D014364:Tryptophan / Q000235:genetics*
D014364:Tryptophan