RNase E-dependent cleavages in the 5' and 3' regions of the Escherichia coli unc mRNA.

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RESUMO

The endonucleolytic processing of the unc mRNA encoding the eight subunits of the Escherichia coli F1F0-ATPase was studied. Northern (RNA) blots of mRNA expressed from a plasmid which contained the 3'-terminal portion of the operon including the uncDC sequences revealed, in addition to the expected 2-kb mRNA, a 0.5-kb RNA species which hybridized to an uncC antisense RNA probe. An uncD antisense RNA probe hybridized to only the 2-kb mRNA, implying that the upstream 1.5-kb fragment is rapidly degraded. The 5' end of the 0.5-kb fragment was determined by primer extension analysis to be 11 bases into the coding region of the uncC gene. In RNase E-deficient strains, the amount of the 0.5-kb product was strongly reduced while the levels of the precursor uncDC transcript remained high. Similar RNase E-dependent processing was found in the chromosomally encoded unc mRNA. As this RNase E-dependent cleavage directly inactivates uncC and appears to leave uncD susceptible to degradation, it seems unlikely to play a role in differential expression of the gene products but may be an important event in unc mRNA degradation. RNase E mutants also showed altered processing of the chromosomally encoded unc mRNA in the uncB region near the 5' end. The expected full-length (7-kb) transcript was recognized when RNA from the RNase E-deficient strain was subjected to Northern blot analysis with uncB- and uncC-specific probes. RNA from strains with functional RNase E lacked the 7-kb transcript but had a 6.2-kb mRNA detectable with the uncC but not the uncB probe. RNase E is therefore implicated in multiple cleavages of the unc mRNA.

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