Stability of an RNA secondary structure affects in vitro transcription pausing in the trp operon leader region

Abstract

Transcription of the tryptophan (trp) operon of Escherichia coli and other bacterial species is regulated by the formation of alternative secondary structures in the leader segment of the transcript. During in vitro transcription of the trp leader region RNA polymerase pauses at base pair 92 after synthesis of an RNA hairpin secondary structure. We studied the dependence of pausing on hairpin stability by examining mutant trp templates containing base pair substitutions in the region corresponding to the hairpin secondary structure. Base changes that lower the stability of the hairpin were found to reduce both the frequency and half-life of RNA polymerase pausing while base changes that do not affect hairpin stability had little effect on pausing. Pausing was enhanced by the nusA protein; this enhancement was greatly reduced on mutant templates specifying less stable hairpins. The frequency of pausing on some mutant templates was correlated with the extent of read-through transcription beyond the trp attenuator, suggesting a possible role for pausing in the coupling of transcription and translation during transcription of the leader region of the operon.