Ribonuclease E provides substrates for ribonuclease P-dependent processing of a polycistronic mRNA.

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 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...

Multiple binding modes of substrate to the catalytic RNA subunit of RNase P from Escherichia coli

“…Ribonuclease P (RNase P) is an essential and ubiquitous ribonucleoprotein enzyme that catalyzes the 59 maturation of all precursor transfer RNAs (ptRNAs)+ Precursors to Escherichia coli 4+5S RNA, tm RNA (10Sa RNA), Salmonella typhimurium his operon mRNA, and several bacteriophage encoded RNAs are also substrates for E. coli RNase P (Bothwell et al+, 1976a(Bothwell et al+, , 1976bGuerrier-Takada et al+, 1988;Mans et al+, 1990;Alifano et al+, 1994;Komine et al+, 1994;Hartmann et al+, 1995)+ E. coli RNase P is composed of a single catalytic RNA (M1 RNA, 377 nt) and a single protein cofactor (C5 protein, 119 amino acids) (Guerrier-Takada et al+, 1983)+ For the E. coli ptRNA substrates, important features for recognition by M1 RNA in vitro include the T stem/loop, the length of the acceptor stem, and the canonical 39 CCA terminus (McClain et al+, 1987;Kirsebom & Vioque, 1996)+ M1 RNA is believed to have the capacity to recognize its varied substrates through multiple binding modes (Guerrier-Takada et al+, 1989;Knap et al+, 1990;Guerrier-Takada & Altman, 1992)+ Such multiple binding modes could reflect an ability of this enzyme to assume more than one conformation on the pathway to an optimally active conformation that would ultimately lead to a cleavage event+ It has been proposed that the catalytic RNA component of Bacillus subtilis RNase P can assume two conformers to which a single substrate can bind, leading to productive enzyme-substrate complexes (Beebe & Fierke, 1994)+ Chemical probing experiments using Fe-EDTA and other methods have noted changes in M1 RNA structure following association with substrate, which could be indicative of an induced-fit type mechanism leading to the formation of a productive complex (Knap et al+, 1990;Guerrier-Takada & Altman, 1993;Ciesiolka et al+, 1994;Westhof et al+, 1996)+ The divalent metal ion and basic protein cofactors of RNase P may have key roles in modulating conformational changes of M1 RNA, both prior to and following substrate binding+ An increase in Mg 2ϩ concentration or the presence of C5 protein can alter the global architecture of M1 RNA (Westhof et al+, 1996)+ Further, deleterious mutations in M1 RNA can be rescued by high, nonphysiological salt concentration or C5 protein, indicative of their ability to assist M1 RNA in structure formation (Lumelsky & Altman, 1988;Gopalan et al+, 1994)+ C5 protein also increases the binding affinity of M1 RNA in a substrate-d...…”

A specific endoribonuclease, RNase P, affects gene expression of polycistronic operon mRNAs

“…In Salmonella, there is one region in an operon mRNA that appears to be cleaved by RNase P (4). In this report, the noncoding intergenic regions and genes coding for proteins (ORFs) in several operons were cloned separately and examined as substrates for RNase P. Several of the intergenic transcripts were substrates for RNase P. These transcripts accumulate at the restrictive temperature in an E. coli mutant strain thermosensitive for RNase P function.…”

Genome-wide search for yeast RNase P substrates reveals role in maturation of intron-encoded box C/D small nucleolar RNAs

“…The protein subunit appears to stabilize the catalytically active conformation of RNase P RNA and assist with substrate binding (4)(5)(6)(7). In addition to pre-tRNAs, bacterial RNase P is known to process several substrates that are proposed to contain tRNA-like structures: 4.5S RNA, tmRNA, viral RNAs, mRNAs, riboswitches, ColE1 replication origin control RNAs, and C4 antisense RNA from phages P1 and P7 (8)(9)(10)(11)(12)(13)(14)(15)(16). The presence of the protein subunit in the RNase P holoenzyme increases the substrate versatility of the enzyme over the RNA enzyme alone (17).…”