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The polycistronic mRNA of the histidine operon is subject to a processing event that generates a rather stable transcript encompassing the five distal cistrons. The molecular mechanisms by which such a transcript is produced were investigated in Escherichia coil strains carrying mutations in several genes for exo-and endonucleases. The experimental approach made use of S1 nuclease protection assays on in vivo synthesized transcripts, site-directed mutagenesis and construction of chimeric plasmids, dissection of the processing reaction by RNA mobility retardation experiments, and in vitro RNA degradation assays with cellular extracts. We have found that processing requires (1) a functional endonuclease E; (2) target site(s) for this activity in the RNA region upstream of the 5' end of the processed transcript that can be substituted by another well-characterized me-dependent cleavage site; (3) efficient translation initiation of the first cistron immediately downstream of the 5' end; and (4) a functional endonuclease P that seems to act on the processing products generated by ribonuclease E. This is the first evidence that ribonuclease P, an essential ribozyme required for the biosynthesis of tRNA, may also be involved in the segmental stabilization of a mRNA.[Key Words: Escherichia coli; Salmonella typhimurium; his operon; mRNA processing; translation; RNase E; RNase P] Received June 2, 1994; revised version accepted October 18, 1994. mRNA decay in bacteria is mediated by the coordinated action of exonucleases and endonucleases (for review, see Petersen 1992). Polynucleotide phosphorylase (PNPase) and ribonuclease II (RNase II) are the two major 3'--* 5' exonucleases involved in mRNA turnover (Donovan and Kushner 1986). Endoribonuclease III (RNase III) does not affect bulk mRNA stability (Babitzke et al. 1993) and is mostly involved in processing of the 30S rRNA precursor even though RNase III can initiate the functional decay of a few specific mRNAs (Court 1993). Endoribonuclease E (RNase E)was initially characterized as the enzyme that processes the precursor of 5S rRNA (Ghora and Apirion 1978). RNase E is the only endoribonuclease implicated thus far in general mRNA turnover (Arraiano et al. 1988;Mudd et al. 1990b;Babitzke and Kushner 1991). Many mRNAs were shown to contain sites whose cleavage was abolished in strains harboring temperature-sensitive rne mutations at the nonpermissive temperature (Kokoska et al. 1990;Mudd et al. 1990a;Gross 1991 1991; Patel and Dunn 1992;Arraiano et al. 1993;Klug 1993;Mudd and Higgins 1993;Carpousis et al. 1994). rne-dependent cleavages might produce relatively stable processed transcripts, such as the T4 gene 32 (Mudd et al. 1988) and the dicF (Faubladier et al. 1990) and the ghX mRNAs (Brunet al. 1990). In different studies ribonuclease P (RNase P) and other nucleases responsible for stable RNA processing did not seem to be involved in mRNA degradation (Deutscher 1988).We have previously identified in Salmonella typhimurium a rather stable 3900-nucleotide-long mRNA molecule...

Post-transcriptional mechanisms operate in regulation of gene expression in bacteria, the amount of a given gene product being also dependent on the inactivation rate of its own message. Moreover, segmental differences in mRNA stability of polycistronic transcripts may be responsible for differential expression of genes clustered in operons. Given the absence of 5' to 3' exoribonucleolytic activities in prokaryotes, both endoribonucleases and 3' to 5' exoribonucleases are involved in chemical decay of mRNA. As the 3' to 5' exoribonucleolytic activities are readily blocked by stem-loop structures which are usual at the 3' ends of bacterial messages, the rate of decay is primarily determined by the rate of the first endonucleolytic cleavage within the transcripts, after which the resulting mRNA intermediates are degraded by the 3' to 5' exoribonucleases. Consequently, the stability of a given transcript is determined by the accessibility of suitable target sites to endonucleolytic activities. A considerable number of bacterial messages decay with a net 5' to 3' directionality. Two different alternative models have been proposed to explain such a finding, the first invoking the presence of functional coupling between degradation and the movement of the ribosomes along the transcripts, the second one implying the existence of a 5' to 3' processive '5' binding nuclease'. The different systems by which these two current models of mRNA decay have been tested will be presented with particular emphasis on polycistronic transcripts.

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