Single base-pair alterations in the Escherichia coli trp operon leader region that relieve transcription termination at the trp attenuator.

Abstract

A set of regulatory mutants defective in transcription termination at the attenuator in the leader region of the E. coli tryptophan (trp) operon was isolated. 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 DNA 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 .cntdot. 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 [.DELTA.G [free energy change] .simeq. -20 kcal (-84 kJ)]. All of the termination-relief mutations destablilize this structure (.DELTA.G .simeq. -9.0 to -10.5 kcal). 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.