The CafA protein required for the 5 '-maturation of 16 S rRNA is a 5 '-end-dependent ribonuclease that has context-dependent broad sequence specificity

The CafA protein, which was initially described as having a role in either Escherichia coil cell division or chromosomal segregation, has recently bee n shown to be required for the maturation of the 5'-end of 16 S rRNA The se quence of CafA is similar to that of the N-terminal ribonucleolytic half of RNase E, an essential E, coil enzyme that has a central role in the proces sing of rRNA and the decay of mRNA and RNAI, the antisense regulator of Col E1-type plasmids, We show here that a highly purified preparation of CafA i s sufficient in vitro for RNA cutting, We detected CafA cleavage of RNAI an d a structured region from the 5'-untranslated region of ompA. mRNA within segments cleavable by RNaseE, but not CafA cleavage of 9 S RNA at its "a" R Nase E site. The latter is consistent with the finding that the generation of 5 S rRNA from its 9 S precursor can be blocked by inactivation of RNase E in cells that are wild type for CafA Interestingly, however, a decanucleo tide corresponding in sequence to the a site of 9 S RNA was cut efficiently indicating that cleavage by CafA is regulated by the context of sites with in structured RNAs, Consistent with this notion is our finding that althoug h 23 S rRNA is stable in vivo, a segment from this RNA is cut efficient by CafA at multiple sites in vitro, We also show that, like RNase E cleavage, the efficiency of cleavage by CafA is dependent on the presence of a monoph osphate group on the 5'-end of the RNA. This finding raises the possibility that the context dependence of cleavage by CafA may be due at least in par t to the separation of a cleavable sequence from the 5'-end of an RNA, Comp arison of the sites surrounding points of CafA cleavage suggests that this enzyme has broad sequence specificity. Together with the knowledge that Caf A can cut RNAI and ompA mRNA in vitro within segments whose cleavage in viv o initiates the decay of these RNAs, this finding suggests that CafA may co ntribute at some point during the decay of many RNAs in E. coli.