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Studies on Ribonuclease E of ESCHERICHIA COLI and its association with the enzyme Polynucleotide phosphorylase Niguma, Kenneth


Messenger RNAs (mRNA) in Escherichia coli are highly labile molecules due to the combined action of a number of exo- and endoribonucleases that orchestrate their degradation. Two of these enzymes, ribonuclease E (RNase E) and polynucleotide phosphorylase (PNPase) have been implicated as key components of a purported rnRNA degradation complex, otherwise known as the "degradosome" (Py etai, Nature 381, 169-172 (1996)). The purpose of these studies was to identify the site of interaction of PNPase with RNase E (Rne). Antibodies were generated against PNPase, initially against fusion proteins expressing two highly antigenic sites predicted to exist in PNPase, and later against a His(6)-PNPase fusion protein. These antibodies, along with a previously generated anti-RNase E antibody, were used to detect Rne or PNPase at various stages during the partial purification of RNase E. Rne and PNPase were found to remain in a stable complex, in association with other unidentified proteins, after several purification steps, and in particular after anion exchange chromatography. Over-expression and partial purification of Rne deletion mutants revealed that loss of the N-terminal portions of Rne did not prevent the mutant from binding PNPase independently, highlighting the importance of the C-terminal portion of Rne in associating with PNPase. Co-chromatography experiments could not determine whether the N-terminal region of Rne bound directly or indirectly to PNPase. A Far-Western experiment, which separates partially purified proteins in cell lysates and assesses their binding individually, demonstrated that derivatives of Rne retaining the C-terminal acidic tail of Rne were competent to bind PNPase. These experiments illustrating the binding of PNPase to the C-terminus of Rne complement the findings that PNPase binding is lost when the Rne C-terminus is missing (Kido etai, J. Bact, 178, 3917- 3925 (1996)).

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