Mutational Analysis of RNA Structures and Sequences Postulated to Affect 3′ Processing of M1 RNA, the RNA Component of Escherichia coli RNase P

Abstract

When the rnpB gene encoding M1 RNA, the RNA component of Escherichia coli RNase P, is transcribed, the primary M1 RNA transcript (pM1 RNA) is produced and subsequently processed at the 3 end to generate the mature M1 RNA. To study features of pM1 RNA thought to be involved in RNA processing, systematic mutations were introduced in sequence elements and secondary structures surrounding the processing site using p23 RNA, a truncated pM1 RNA transcribed from the internally deleted rnpB gene, as a model substrate and the processing of its mutant derivatives was analyzed in vivo and in vitro. Neither the alteration of two bases forming the processing site nor the disruption of secondary structures surrounding the site significantly affected the processing efficiency although the secondary structures were required for maintaining RNA stability. In contrast, mutations at the rne-dependent site, GAUUU, immediately 3 to the processing site inhibited the processing and the extent of the inhibition varied with the altered sequences. Furthermore, the processing of the mutants of the rne-dependent site as well as wild-type p23 RNA was inhibited in an E. coli rne ts strain at the nonpermissive temperature.Ribonuclease P (RNase P) is a processing enzyme that catalyzes the endonucleolytic removal of 5Ј leader sequences from precursors of tRNAs to generate the mature 5Ј ends of tRNAs (1). In Escherichia coli, RNase P is composed of the RNA subunit (M1 RNA, 377 nucleotides) and the protein subunit (C5 protein, 119 amino acid residues) (2-4).The promoter region of the rnpB gene coding for M1 RNA contains three regions of homology to the E. coli consensus promoter sequence (5). In vitro and in vivo transcriptions arise from the nearest promoter, P-1, almost exclusively (6), even though transcripts can originate from the upstream promoters (5). The 3Ј-flanking region of the rnpB gene contains a repeated DNA sequence of 113 base pairs. This sequence, which includes both the 3Ј terminal 24 nucleotides of the M1 RNA coding sequence and the region for transcription termination, is repeated almost 3.5 times (7). The region for transcription termination contains a stem and loop structure characteristic of structures found near regions of -independent transcription termination (8), and functions in a -independent fashion in vitro (9). In vivo studies using the galactokinase transcription fusion plasmids (10) indicate that more than 94% of transcription is terminated at the first terminator, T1, in the first repeating unit. Therefore, the RNA product initiated from P-1 and terminated at T1 should be a major primary transcript of the rnpB gene from which mature M1 RNA is derived. This transcript, termed pM1 RNA (primary transcript), carries extra stretches of 36 nucleotides containing a termination stem and loop at the 3Ј end of M1 RNA which are removed by a processing event. pM1 RNA of 413 nucleotides is processed to form mature M1 RNA in vitro by an S30 extract (9). On the other hand, a 40% ammonium sulfate precipitate of S200 extract from...

Novel function of C5 protein as a metabolic stabilizer of M1 RNA

“…The E. coli temperature-sensitive C5 protein mutant isogenic strains NHY312 (rnpA + ) and NHY322 (rnpA49) [21] were also used for RNA analysis. Plasmid pLM1, a derivative of the pGEM3 vector, which contains the rnpB sequence with flanking sequences spanning positions -270 to +1286 [23], was used to construct additional plasmids capable of overexpressing truncated M1 RNAs. The deletions were introduced by gene splicing via overlap extension (SOEing) PCR mutagenesis, as previously described [24], to generate plasmids, pLMdP3, pLMdP8/9, and pLMdP12.…”

Identification of Amino Acid Residues in the Catalytic Domain of RNase E Essential for Survival of Escherichia coli: Functional Analysis of DNase I Subdomain

“…Although the functional role of this region has not been previously identified, mutations in the region could in principle interfere with the ribonucleolytic activity of RNase E by either reducing binding to the RNA substrate or inhibiting the catalytic activity of the enzyme. To further understand the basis for the loss of ribonucleolytic activity of these mutant proteins, we tested the ability of N-Rne-NC proteins mutated in the DNase I subdomain (A326T and L385P) to bind to p23 RNA, which is a truncated pM1 RNA that is processed by RNase E to a product termed 23 RNA (Kim et al 1996). Gel shift assays indicated the inability of these N-Rne-NC mutants to bind to p23 RNA ( Figure 4B).…”

Expression of a Small Protein Encoded by the 3' Flanking Sequence of the Escherichia coli rnpB Gene

“…coli cells containing pLMd23, as described previously. 16 RNA samples were electrophoresed on a 5% polyacrylamide gel containing 7 M urea, electrotransferred to a Hybond-N + membrane (Amersham), and hybridized. 21 The probe employed was antisense M1 RNA generated from HindIIIlinearized pLMd23 DNA using T7 RNA polymerase and [α-…”

“…Plasmid pLMd23 was a derivative of pLM1 with an internal deletion between positions +57 and +330 in the M1 RNA structural region. 16 The pGEX4T-1 plasmid (Amersham Biosciences) was used as a PCR template for amplifying the GST coding sequence.…”

“…14,15 The majority of transcription occurs from the nearest promoter, P-1, with initiation at position 1 of mature M1 RNA, and terminates at an intrinsic terminator (T1) to generate precursor M1 RNA (pM1 RNA) containing an extra 36 nucleotide stretch. 16,17 The 3' flanking region of rnpB contains 113 bp repeats that are not perfectly identical, including the sequence coding for the 3' terminal 24 nucleotides of M1 RNA and the intrinsic transcription termination region. [18][19][20] This unit begins from position +354, and is successively repeated almost 3.5 times.…”