In Escherichia coli, the RNA degradosome is a protein complex involved in the general degradation of mRNA and in post-transcriptional gene regulation. The principal components of the degradosome complex are the endoribonuclease RNase E, the phosphorolytic exoribonuclease PNPase, the ATP-dependent RNA helicase RhlB, and the glycolytic enzyme enolase. The RNase E protein is a 1061 amino acid protein which can be divided into three major functional portions: the N-terminal catalytic activity portion; the central membrane anchoring and RNA binding portion; and the C terminal protein-interaction portion which bind to other major degradosome components. RraA and RraB (Regulator of RNase E activity) are protein regulators of RNase E discovered in our lab, which regulate RNase E by binding to the RNase E C-terminal region. The work presented here describes the regulation of rraB gene expression and in vitro studies of degradosome assembly and the effects of RraA/RraB inhibition. rraB is transcribed from its own promoter PrraB. A transposon insertion into glmS encoding glucosamine-6P synthase resulted in a 4 fold increase in the PrraB activity from a PrraB-lacZ fusion the indicating that glmS is serves as a negative regulator of rraB transcription. Consistent with this discovery, real-time RT-PCR revealed that glmS::Tn5 results in a 5-fold increase on the steady-state level of rraB mRNA. As part of this work we have reconstituted the degradosome from individually purified proteins. The binding sites of RraA and RraB overlap with the RNA binding and the RhlB interaction sites within the C-terminus of RNase E. We have characterized the effects of RraA and RraB on the decay of various RNA substrates by reconstituted degradosomes: RraA and RraB proteins were shown to inhibit the hydrolysis reaction a short substrate by RNase E by up to 50% in a mixed inhibition pattern. Inhibition of the decay of the long RNA substrates RNA1 or dsbC was much more severe with the RNA processing activity becoming reduced by as much as 80%. These studies have delineated the kinetic consequences of inhibition by RraA and RraB and provide further insights into the mechanisms that control RNA decay in bacteria. / text

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