Mechanistic analysis of selective inhibition of RNA processing in Escherichia coli



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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