Maturation of Escherichia coli tryptophan operon mRNA: evidence for 3' exonucleolytic processing after rho-dependent termination

Abstract

The mature 3' end of Escherichia coli tryptophan operon mRNA in vivo coincides with a site (trp t) having features commonly associated with rho-independent terminators in bacteria. Efficient generation of this 3' end in vivo is nevertheless affected by a distal rho-dependent site (trP-t'), though these two sites behave independently in vitro. We have cloned these sites upstream of the galactokinase gene (galK), and galactokinase levels in vivo indicate that, as terminators per se, their efficiencies (37% for trp t, and 79% for trp t') do not differ significantly from those observed in vitro. However, when the trp t hairpin is placed between galK and a downstream copy of trp t', galactokinase levels are enhanced 2- to 3-fold. This suggests the involvement of a post-transcriptional event, such as RNA processing, in determining the level of gene activity. Indeed, in the presence of the 3' exonuclease RNase II, mRNA terminated by rho factor in vitro at the trp t' site is processed back to the trp t site. The remote trp t' region appears to be the major termination site for trp mRNA, and the trp t hairpin serves a dual function-as a minor terminator, and as a protective barrier to 3' exonucleolytic degradation. We infer that the tandem terminators, rho factor, and RNA processing are all required to generate the mature 3' end of this bacterial mRNA.