Partial characterization of an RNA component that copurifies with Saccharomyces cerevisiae RNase P.

Abstract

Saccharomyces cerevisiae cellular RNase P is composed of both protein and RNA components that are essential for activity. The isolated holoenzyme contains a highly structured RNA of 369 nucleotides that has extensive sequence similarities to the 286-nucleotide RNA associated with Schizosaccharomyces pombe RNase P but bears little resemblance to the analogous RNA sequences in procaryotes or S. cerevisiae mitochondria. Even so, the predicted secondary structure of S. cerevisiae RNA is strikingly similar to the bacterial phylogenetic consensus rather than to previously predicted structures of other eucaryotic RNase P RNAs.Mature 5' termini of tRNAs in both procaryotes and eucaryotes are generated by cleavage with a structurespecific endonuclease, RNase P. Extensive study of RNase P activities from Escherichia coli and Bacillus siubtilis has shown that both enzymes consist of single RNA and protein species (14,16,19,21,25,[29][30][31]38,43,44,51). Although temperature-sensitive mutations in these individual components have shown that both are required for RNase P activity in vivo (24,25,31,38,41,46,47), each RNA component alone can recognize and cleave substrates under some conditions in vitro (2,16,19,20,44). The enzymic RNAs can form extensively base-paired secondary structures as judged by theoretical calculations and cleavage sensitivity structure analyses (3,17,43,44). Extensive phylogenetic sequence comparisons among the bacterial RNAs suggest several short domains of conserved sequences and a conserved secondary structure (26). This structure, rather than Watson-Crick base pairing with the substrates, is suspected of being primarily responsible for recognition of pre-tRNAs, since several RNA structural disruptions have been shown to reduce catalytic activity. A sequence originally thought to contribute to substrate recognition through complementarity to the invariant T4JCG sequence in tRNAs is dispensable (18,34).RNase P RNA activities have been reported in a number of eucaryotes (1,5,6,8,11,13,22,28,32,33,35,36), but there has been significant disagreement as to the nature of the enzymes. In particular, the RNase P from Xenopis laevis has been reported to be insensitive to micrococcal nuclease and found as part of a large polypeptide complex (6). For all other nuclear and mitochondrial enzymes tested, RNA components have been shown to be essential on the basis of either nuclease sensitivity of the enzyme or genetic evidence. Other than one report on HeLa cell RNase P (15), the ability to denature and reconstitute the eucaryotic components has not been reported and there have been no instances of RNA components having catalytic activity in the absence of protein (39). RNA subunits essential for the activity of the enzyme (28, 37) in SchiZosaccharomnvces pombe and Saccharomvces cerev'isiae mitochondria have been identified and their sequences have been determined (33,37). Surprisingly, neither the nucleotide sequences nor the predicted secondary structures of these RNAs bear any striking resemblance to each o...

Characterization and partial purification of two pre‐tRNA 5′‐processing activities from Daucus carrota (carrot) suspension cells

“…The sensitivity of both of these activities to treatment by micrococcal nuclease is in agreement with many other RNase P studies (Doerson et al, 1985;Krupp et aL, 1986;Bartkiewicz et al, 1989;Lee and Engelke, 1989;Doria et al, 1991) (Figure 7). Most RNase P activities examined thus far, whether prokaryotic or eukaryotic (both nuclear and organellar), have essential RNA components.…”

“…In most instances, the RNA species that have been studied co-purify with RNase P activity and it can be shown that the holoenzyme is sensitive to treatment with micrococcal nuclease (Bartkiewicz et al, 1989;Doerson et al, 1985;Doria et al, 1991;Krupp eta/., 1986;Lee and Engelke, 1989). More direct evidence for the role of an RNA component in eukaryotes can be found in the case of the human nuclear RNase P, which can be immunoprecipitated by antibodies against the protein moiety of the E. colienzyme.…”

Evidence for an RNA-based catalytic mechanism in eukaryotic nuclear ribonuclease P

“…Transfer RNA molecules are synthesized in vivo as precursors (pre-tRNA) with extra sequences at their 59 and 39 termini+ These precursor-specific RNAs must be removed before the tRNA can function+ Ribonuclease P (RNase P) is the enzyme responsible for removing the 59 extension, or leader, of precursor transfer RNAs in all organisms+ It hydrolyzes the phosphodiester bond 59 to the first nucleotide of the tRNA domain, releasing a 59 leader with a 39 hydroxyl group, and a mature tRNA with a 59 phosphoryl+ A remarkable feature of this enzyme is its composition: the RNA component of bacterial RNase P is the catalytic moiety (Guerrier-Takada et al+, 1983;reviewed in Frank & Pace, 1998), with the protein subunit assisting in maintaining the structure of the RNA subunit and facilitating discrimination between substrate and product (Reich et al+, 1988;Tallsjo & Kirsebom, 1993;Kurz et al+, 1998)+ Recently, the complete polypeptide composition of yeast RNase P (Chamberlain et al+, 1998) and the nearly complete composition of the human enzyme (Jarrous et al+, 1998) have been determined+ The yeast enzyme contains at least nine polypeptides, ranging from ;16 kDa to 100-115 kDa+ As predicted, only about 20% of the eukaryotic holoenzyme mass is RNA+ In contrast, the bacterial holoenzyme is 80-90% RNA+ In Saccharomyces cerevisiae nuclear RNase P, the RNA subunit is essential for activity both in vitro and in vivo (Lee & Engelke, 1989;Lee et al+, 1991), but has not been shown to be directly involved in catalysis+ The current structural and phylogenetic evidence (Tranguch & Engelke, 1993;Chen & Pace, 1997;Pitulle et al+, 1998;reviewed in Frank & Pace, 1998) supports the hypothesis that eukaryotic RNase P RNA possesses much or all of the catalytic machinery and that the role of the protein is to maintain the required tertiary structure of the RNA+ To determine whether the reaction mechanism of a eukaryotic ribonucleoprotein RNase P was similar to that of the bacterial ribozyme RNase P, we analyzed the ability of S. cerevisiae nuclear RNase P to cleave a pre-tRNA containing a sulfur substitution at the pro-R P nonbridging oxygen of the scissile bond+ The bacterial RNase P RNA subunit requires divalent metal cations, preferably Mg(II) or Mn(II), for substrate binding and catalysis (Gardiner et al+, 1985;Guerrier-Takada et al+, 1986;Smith et al+, 1992;Beebe et al+, 1996)+ Using the Escherichia coli RNase P RNA subunit and a phosphorothioate-substituted pre-tRNA, previous experiments revealed that the catalytic RNA subunit absolutely requires a Mg 2ϩ ion coordinated to the pro-R P nonbridging oxygen of the scissile bond (Chen et al+, 1997)+ As a consequence, bacterial RNase P cannot cl...…”

The RNA component of RNase P in Schizosaccharomyces species

“…[12]). Furthermore, none of the RNAs from the eukaryotic enzymes identified to date have been shown to be catalytic in vitro, the hallmark of eubacterial RNase P [1,11,13,14]. ty copurifies with two RNAs, designated Kl-and K2-RN&.…”