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Ribonuclease E provides substrates for ribonuclease P-dependent processing of a polycistronic mRNA
P Alifano1, F Rivellini, C Piscitelli
1Dipartimento di Biologia e Patologia Cellulare e Molecolare L. Califano, Università degli Studi di Napoli, Italy.
Abstract:
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 coli 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 rne-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.
Insights
This study reveals that bacterial mRNA processing requires specific endonucleases, including ribonuclease E and ribonuclease P, to stabilize transcripts. Efficient translation initiation is also crucial for this mRNA maturation process.
Area of Science:
- Molecular Biology
- Microbiology
- Genetics
Background:
- Polycistronic mRNAs, like that of the histidine operon, undergo processing to yield stable transcripts.
- The precise molecular mechanisms governing this mRNA processing and stabilization remain incompletely understood.
Purpose of the Study:
- To elucidate the molecular mechanisms underlying the processing and stabilization of the polycistronic histidine operon mRNA in Escherichia coli.
- To identify the specific endonucleases and RNA features involved in generating a stable mRNA transcript.
Main Methods:
- Utilized S1 nuclease protection assays to analyze in vivo synthesized transcripts.
- Employed site-directed mutagenesis and chimeric plasmid construction to investigate RNA processing.
- Performed RNA mobility retardation experiments and in vitro RNA degradation assays with cellular extracts.
Main Results:
- Identified functional endonuclease E and specific target sites upstream of the processed transcript as critical for processing.
- Demonstrated that efficient translation initiation of the first cistron is essential.
- Found that functional endonuclease P acts on ribonuclease E processing products, contributing to stabilization.
Conclusions:
- The processing and segmental stabilization of histidine operon mRNA depend on a coordinated action of endonucleases E and P.
- This study provides the first evidence for ribonuclease P's involvement in mRNA stabilization, extending its known functions beyond tRNA biosynthesis.