Roles of polyadenylation and nucleolytic cleavage in the filamentous phage mRNA processing and decay pathways in Escherichia coli.

To define basic features of mRNA processing and decay in Escherichia coli, we have examined a set of mRNAs encoded by the filamentous phage f1 that have structures typical of bacterial mRNAs. They bear a stable hairpin stem-loop on the 3' end left from rho-independent termination and are known to undergo processing by RNase E. A small percentage of the f1 mRNAs were found to bear poly(A) tails that were attached to heterogeneous positions near the common 3' end. In a poly(A) polymerase-deficient host, the later-appearing processed mRNAs were stabilized, and a novel small RNA accumulated. This approximately 125-nt RNA proved to arise via RNase E cleavage from the 3'-terminal region of the mRNAs bearing the terminator. Normally ribosomes translating gene VIII appear to protect this cleavage site from RNase E, so that release of the fragment from the mRNAs occurs very slowly. The data presented define additional steps in the f1 mRNA processing and decay pathways and clarify how features of the pathways are used in establishing and maintaining the persistent filamentous phage infection. Although the primary mode of decay is endonucleolytic cleavage generating a characteristic 5' --> 3' wave of products, polyadenylation is involved in part in degradation of the processed mRNAs and is required for turnover of the 125-nt mRNA fragment. The results place polyadenylation at a later rather than an initiating step of decay. They also provide a clear illustration of how stably structured RNA 3' ends act as barriers to 3' --> 5' exonucleolytic mRNA decay.     

The regulatory protein RraA modulates RNA-binding and helicase activities of the E. coli RNA degradosome

The Escherichia coli endoribonuclease RNase E is an essential enzyme having key roles in mRNA turnover and the processing of several structured RNA precursors, and it provides the scaffold to assemble the multienzyme RNA degradosome. The activity of RNase E is inhibited by the protein RraA, which can interact with the ribonuclease''s degradosome-scaffolding domain. Here, we report that RraA can bind to the RNA helicase component of the degradosome (RhlB) and the two RNA-binding sites in the degradosome-scaffolding domain of RNase E. In the presence of ATP, the helicase can facilitate the exchange of RraA for RNA stably bound to the degradosome. Our data suggest that RraA can affect multiple components of the RNA degradosome in a dynamic, energy-dependent equilibrium. The multidentate interactions of RraA impede the RNA-binding and ribonuclease activities of the degradosome and may result in complex modulation and rerouting of degradosome activity.