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Endonuclease processing in the rif region of the Escherichia coli chromosome

University of British Columbia

Endonuclease processing of mRNA derived from the rif region near 90 minutes on the Escherichia coli chromosome was examined. This region contains the secE-nusG and the rplKAJL-rpoBC (ribosomal protein) operons and it is thought that endonuclease processing by RNase E and RNase III plays some role in translational regulation and/or degradation of these transcripts. The purpose of this study was to investigate the effect that RNase E and RNase III cleavage had on the stability of transcripts derived from the rif region. Several RNase E and RNase III cleavage sites were identified in the secE-nusG and ribosomal protein operons. Total RNA was isolated from wild type and RNase Ets or RNase III deficient E. coli strains and hybridized to DNA probes from the rif region. By S1 nuclease protection assays, cleavage by RNase E and RNase III could be designated to those sites which were not cleaved in the RNase Ets (nonpermissive temperature) or RNase III deficient strains. The secE-nusG leader region contains an RNase III site and has the potential to form a secondary structure characteristic of other known substrates of RNase III. An RNase E site identified in the L1-L10 intergenic space shares striking sequence and structural similarity with the RNase E consensus sequence and downstream stem loop thought to be involved in RNase E recognition. As well, another processing site was observed in the L1-L10 intergenic space which may be a result of RNase III activity or processing by an unidentified nuclease. The effect of RNase E and RNase III processing on the stability of the secE-nusG and ribosomal protein transcripts was studied. By S1 nuclease mapping, the decay of transcripts was monitored at two minute intervals in rifampicin-treated E. coli cultures that either had a temperature-sensitive RNase E mutation or was deficient in RNase III activity. When the RNase Ets strain was examined at the permissive temperature, the transcripts decayed normally with half-lives resembling those seen in the isogenic wild type parent. But when the strain was shifted to the restricted temperature, all the transcripts probed decayed more slowly, by a factor greater than 10-fold. In the absence of RNase III activity, only a slight stabilization of the transcripts was observed. The results suggest that RNase E cleavage plays a significant role in the degradation of the secE-nusG and ribosomal protein transcripts either as the rate determining step initiating decay and/or as a secondary step that indirectly influences decay. Furthermore, and perhaps most importantly, these observations can be used to demonstrate that the synthesis rates of these mRNAs are severely reduced when their degradation mediated by RNase E, and to a lesser extent, RNase III, is prevented. That is, the increase in the mRNA half-life is almost exactly compensated by a concomitant reduction in the mRNA synthesis rate such that the amount of mRNA remains virtually constant. How amount, half-life, and synthesis rates of mRNA are related is unknown.

Thesis/Dissertation

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2008-08-07

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http://hdl.handle.net/2429/1304

Biochemistry and Molecular Biology

University of British Columbia

UBCV

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