RIBONUCLEASE-E PROVIDES SUBSTRATES FOR RIBONUCLEASE P-DEPENDENT PROCESSING OF A POLYCISTRONIC MESSENGER-RNA

The polycistronic mRNA of the histidine operon is subject to a process ing event that generates a rather stable transcript encompassing the f ive distal cistrons. The molecular mechanisms by which such a transcri pt is produced were investigated in Escherichia coli strains carrying mutations in several genes for exo- and endonucleases. The experimenta l approach made use of S1 nuclease protection assays on in vivo synthe sized transcripts, site-directed mutagenesis and construction of chime ric plasmids, dissection of the professing reaction by RNA mobility re tardation experiments, and in vitro RNA degradation assays with cellul ar extracts. We have found that processing requires (1) a functional e ndonuclease E; (2) target site(s) for this activity in the RNA region upstream of the 5' end of the processed transcript that can be substit uted by another well-characterized rne-dependent cleavage site; (3) ef ficient translation initiation of the first cistron immediately downst ream of the 5' end; and (4) a functional endonuclease P that seems to act on the processing products generated by ribonuclease E. This is th e first evidence that ribonuclease P, an essential ribozyme required f or the biosynthesis of tRNA, may also be involved in the segmental sta bilization of a mRNA.