Identification of a precursor molecular for the RNA moiety of the processing enzyme RNase P.

Gurevitz, Michael; Jain, Swatantra Kumar; Apirion, David

doi:10.1073/pnas.80.14.4450

Cited by 45 publications

(39 citation statements)

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“…DNA sequencing of the PCR product of the rnpB gene encoding M1 RNA indicated that the sequence of the gene downstream of the mature 3Ј terminus in the mutant is GATTT, the same as that published previously (14,15), and the same as the wild-type precursor sequence (Table 2). Thus, we conclude that the A residues in the third and fourth positions must be added posttranscriptionally.…”

Section: Methodsmentioning

confidence: 51%

“…However, products were found to accumulate in some exoribonuclease-deficient strains that contained as many as 10 extra nucleotides at their 3Ј ends (8). Likewise, in a recent study of the maturation of M1 RNA, the catalytic subunit of RNase P, products with up to six additional 3Ј residues were observed (9), despite the fact that RNase E is thought to cleave this precursor at a position only one or two nucleotides downstream from the mature 3Ј terminus (14,15).…”

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confidence: 99%

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Polyadenylation of stable RNA precursors in vivo

Pandit

Deutscher

1998

Proc. Natl. Acad. Sci. U.S.A.

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Polyadenylation at the 3 terminus has long been considered a specific feature of mRNA and a few other unstable RNA species. Here we show that stable RNAs in Escherichia coli can be polyadenylated as well. RNA molecules with poly(A) tails are the major products that accumulate for essentially all stable RNA precursors when RNA maturation is slowed because of the absence of processing exoribonucleases; poly(A) tails vary from one to seven residues in length. The polyadenylation process depends on the presence of poly(A) polymerase I. A stochastic competition between the exoribonucleases and poly(A) polymerase is proposed to explain the accumulation of polyadenylated RNAs. These data indicate that polyadenylation is not unique to mRNA, and its widespread occurrence suggests that it serves a more general function in RNA metabolism.Cellular RNA molecules have long been divided into two groups. The first group, the stable RNAs, includes mainly rRNA, tRNA, and a variety of other small RNAs, and represents Ͼ95% of the total cellular RNA population. These RNAs have long lifetimes relative to the generation times of the cells in which they reside. The second group, unstable RNAs, consists primarily of mRNAs, which are a small fraction of the total RNA population and have half-lives that are usually much shorter than the generation time of the cell. The unstable RNAs generally contain a poly(A) tract at their 3Ј ends that contributes to their turnover; for mRNAs, poly(A) contributes to their role in translation (1-5). However, polyadenylation of stable RNAs has rarely been seen, and has not been considered important for stable RNA metabolism.In previous studies of the maturation of tRNA (6, 7), 5S RNA (8), and other small, stable RNAs (4.5S, 6S, M1, and tmRNA) (9) in Escherichia coli, it was shown that exoribonucleolytic trimming was a necessary step in the formation of the 3Ј termini of these various molecules. Thus, in the absence of the requisite 3Ј to 5Ј exoribonucleases, precursor products with extra nucleotides at their 3Ј ends accumulated. In some instances, however, these extra sequences were longer than expected. For example, 5S RNA is known to be released from long rRNA transcripts by RNase E endonucleolytic cleavages that leave three additional residues at each end of the processing intermediate (10-13). However, products were found to accumulate in some exoribonuclease-deficient strains that contained as many as 10 extra nucleotides at their 3Ј ends (8). Likewise, in a recent study of the maturation of M1 RNA, the catalytic subunit of RNase P, products with up to six additional 3Ј residues were observed (9), despite the fact that RNase E is thought to cleave this precursor at a position only one or two nucleotides downstream from the mature 3Ј terminus (14,15).In this paper we provide an explanation for these unexpected observations. To do so, we determined the nucleotide sequences at the 3Ј ends of the 5S and M1 RNA products that accumulate in the multiple exoribonuclease-deficient cells. This was acco...

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Section: Methodsmentioning

confidence: 51%

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confidence: 99%

Polyadenylation of stable RNA precursors in vivo

Pandit

Deutscher

1998

Proc. Natl. Acad. Sci. U.S.A.

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“…However, some of the rne-dependent sites listed in Table 1 must be considered to be only putative sites because cleavage by RNase E in vitro has not been demonstrated. A few rae-dependent sites are apparently not cleaved by partially purified RNase E Gurevitz et al 1983;Nilsson et al 1988;Subbarao and Apirion 1989) but by other activities like RNase K (Lunberg et al 1990), RNase F (Gurevitz et al 1982), or RNase 111 (Srivastava et al 1990). …”

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confidence: 99%

Specificity of Escherichia coli endoribonuclease RNase E: in vivo and in vitro analysis of mutants in a bacteriophage T4 mRNA processing site.

Ehretsmann¹,

Carpousis²,

Krisch³

1992

Genes Dev.

209

147

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Endoribonuclease RNase £ has an important role in the processing and degradation of bacteriophage T4 and Escherichia coli mRNAs. We have undertaken a mutational analysis of the -71 RNase E processing site of T4 gene 32. A Series of mutations were introduced into a synthetic T4 sequence cloned on a plasmid, and their effects on processing were analyzed in vivo. The same mutations were transferred into T4 by homologous recombination. In both the plasmid and the phage contexts the processing of the transcripts was similarly affected by the mutations. Partially purified RNase E has also been used to ascertain the effect of these mutations on RNase E processing in vitro. The hierarchy of the efficiency of processing of the various mutant transcripts was the same in vivo and in vitro. These results and an analysis of all of the known putative RNase E sites suggest a consensus sequence RAUUW (R = A or G; W ^ A or U) at the cleavage site. Modifications of the stem-loop structure downstream of the -71 site indicate that a secondary structure is required for RNase E processing. Processing by RNase E was apparently inhibited by sequences that sequester the site in secondary structure.

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“…5). Multiple transcripts arising from any of three promoters can be processed at the 3' end to remove a 38-nucleotide terminator sequence (13,14,28 (13). An RNA processing event which removes the terminator stem and loop could remove this messenger sequence from the nascent Mi RNA in unterminated RNA.…”

Section: Resultsmentioning

confidence: 99%

Tandem promoters preceding the gene for the M1 RNA component of Escherichia coli ribonuclease P.

Motamedi¹,

Lee²,

Schmidt³

1984

Proc. Natl. Acad. Sci. U.S.A.

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The In an effort to address the question of ribonuclease P regulation, we determined the sequence of the promoter region and structural DNA of a cloned gene for the RNA component, Ml RNA, and examined its transcription. Although three regions are observed with substantial homology to the consensus E. coli promoter sequence (3, 4), in vitro transcription arose from the nearest promoter exclusively. Analysis of Ml RNA transcription products by Si nuclease mapping, however, indicates that transcripts from all three promoter sequences are present in vivo. These transcripts are apparently processed in a multistep pathway. EXPERIMENTAL PROCEDURESBacterial and Phage Strains and Plasmids. Plasmid pLN2(2) was grown in E. coli A49 (5) and purified by CsCl/ethidium bromide centrifugation. E. coli strain JM101 (6) was from the collection of J. Robbins. M13mp7 phage (6) and replicative form DNA were from Bethesda Research Laboratories. Fig. 1 were done in triplicate. RNA fingerprinting of the in vitro transcript of Ml RNA was done as described by Brownlee (9).In Vitro Transcription. The reaction conditions were similar to those described by Farnham and Platt (10) Nucleotides, salts, and DNA were mixed together and preincubated for 5 min at 370C. E. coli RNA polymerase (1 ,tg) was then added to initiate the reaction. The reaction mixture was incubated for 20 min at 370C. The reaction was stopped by adding an equal volume of stop solution containing 0.8% NaDodSO4 and 160 mM EDTA. Carrier tRNA (20 ,ug) was added, and the products then were extracted with redistilled, water-saturated phenol. Sodium acetate was added to 0.3 M final concentration, and RNAs were precipitated by addition of 3 vol of absolute ethanol. The RNA pellet was washed once with 70% ethanol/0.2 M potassium acetate and was dried briefly under vacuum. The isolated RNA was resuspended in 20 tkl of the dye mixture, which contained 10 M urea, 0.5% xylene cyanol FF, and 0.5% bromophenol blue. The isolated transcripts were analyzed by electrophoresis in a 5% polyacrylamide/Tris/borate/EDTA gel in 7 M urea which contained 0.1% NaDodSO4. S1 Nuclease Mapping. The 480-bp Sau3Al fragment of pLN2 encoding the 5' end of the Ml RNA gene was labeled at the 5' ends with [y-32P]ATP and polynucleotide kinase.Abbreviation: bp, base pair(s).

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Identification of a precursor molecular for the RNA moiety of the processing enzyme RNase P.

Cited by 45 publications

References 38 publications

Polyadenylation of stable RNA precursors in vivo

Polyadenylation of stable RNA precursors in vivo

Specificity of Escherichia coli endoribonuclease RNase E: in vivo and in vitro analysis of mutants in a bacteriophage T4 mRNA processing site.

Tandem promoters preceding the gene for the M1 RNA component of Escherichia coli ribonuclease P.

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