Extensive deproteinization of Dictyostelium discoideum RNase P reveals a new catalytic activity.

Nuclear Dictyostelium discoideum RNase P was subjected to vigorous deproteinization procedures. After treatment with proteinase K followed by phenol extraction of samples containing D. discoideum RNase P activity, a new enzymatic activity was recovered. The proteinase K/phenol/SDS treated enzyme cleaves Schizossacharomyces pombe tRNAser (supS1), D. discoideum tRNASer and tRNALeu precursors several nucleotides upstream of the cleavage site of RNase P, liberating products with 5'-hydroxyl ends. This activity seems to be associated with one or two RNA molecules copurifying with D. discoideum RNase P activity as judged by its inhibition in the presence of micrococcal nuclease, which is in contrast to its resistance to proteinase K/phenol/SDS treatment.     

The Schizosaccharomyces pombe sup3-i suppressor recognizes ochre, but not amber codons in vitro and in vivo.

The inefficient suppressor sup3-i of the fission yeast Schizosaccharomyces pombe is an ochre suppressor. Sup3-i was derived from the efficient serine inserting UGA suppressor sup3-e. The cloning and sequencing of the sup3-i gene indicate that the suppressor is different from the parent sup3-e by a C----T substitution in the sequence coding for the middle position of the anticodon. In vitro translation assays supplemented with purified sup3-i tRNA and programmed with Xenopus globin mRNAs lead to the accumulation of a readthrough product in response to UAA termination signals, but not in response to UGA termination codons. Transformation of Saccharomyces cerevisiae nonsense mutant strains with plasmid DNA carrying the S. pombe sup3-i gene, led to ochre, but not amber or UGA suppression in vivo.     

The nucleotide sequence of a UGA suppressor serine tRNA from Schizosaccharomyces pombe.

The UGA suppressor tRNA produced by Schizosaccharomyces pombe strain sup3-e was purified to homogeneity. It can be aminoacylated with a serine by a crude aminoacyl-tRNA synthetase preparation from S. pombe cells. By combining post-labeling fingerprinting and gel sequencing methods the nucleotide sequence of this tRNA was determined to be: pG-U-C-A-C-U-A-U-G-U-C-ac4C-G-A-G-D-G-G-D-D-A-A-G-G-A-m2G2-psi-U-A-G-A-N-U-U-C-A-i6A-A-psi-C-U-A-A-U-G-G-G-C-U-U-U-G-C-C-C-G-m5C-G-G-C-A-G-G-T-psi-C-A-m1A-A-U-C-C-U-G-C-U-G-G-U-G-A-C-G-C-C-A OH. The anticodon sequence u ca is complementary to the UGA codon.     

Kinetics of the processing of the precursor to 4.5 S RNA, a naturally occurring substrate for RNase P from Escherichia coli.

A study was made of the cleavage by M1 RNA and RNase P of a non-tRNA precursor that can serve as a substrate for RNase P from Escherichia coli, namely, the precursor to 4.5 S RNA (p4.5S). The overall efficiency of cleavage of p4.5S by RNase P is similar to that of wild-type tRNA precursors. However, unlike the reaction with wild-type tRNA precursors, the reaction catalyzed by the holoenzyme with p4.5S as substrate has a much lower Km value than that catalyzed by M1 RNA with the same substrate, indicating that the protein subunit plays a crucial role in the recognition of p4.5S. A model hairpin substrate, based on the sequence of p4.5S, is cleaved with greater efficiency than the parent molecule. The 3'-terminal CCC sequence of p4.5 S may be as important for cleavage of this substrate as the 3'-terminal CCA sequence is for cleavage of tRNA precursors.     

Conditional expression of RNase P in the cyanobacterium Synechocystis sp. PCC6803 allows detection of precursor RNAs. Insight in the in vivo maturation pathway of transfer and other stable RNAs

We have constructed a strain (CT1) that expresses RNase P conditionally with the aim to analyze the in vivo tRNA processing pathway and the biological role that RNase P plays in Synechocystis 6803. In this strain, the rnpB gene, coding for the RNA subunit of RNase P, has been placed under the control of the petJ gene promoter (P(petJ)), which is repressed by copper, cell growth, and accumulation of RNase P RNA is inhibited in CT1 after the addition of copper, indicating that the regulation by copper is maintained in the chimerical P(petJ)-rnpB gene and that RNase P is essential for growth in Synechocystis. We have analyzed several RNAs by Northern blot and primer extension in CT1. Upon addition of copper to the culture medium, precursors of the mature tRNAs are detected. Furthermore, our results indicate that there is a preferred order in the action of RNase P when it processes a dimeric tRNA precursor. The precursors detected are 3'-processed, indicating that 3' processing can occur before 5' processing by RNase P. The size of the precursors suggests that the terminal CCA sequence is already present before RNase P processing. We have also analyzed other potential RNase P substrates, such as the precursors of tmRNA and 4.5 S RNA. In both cases, accumulation of larger than mature size RNAs is observed after transferring the cells to a copper-containing medium.     

Processing of histidine transfer RNA precursors. Abnormal cleavage site for RNase P

The 5'-terminal guanylate residue (G-1) of mature Escherichia coli tRNA(His) is generated as a result of an unusual cleavage by RNase P (Orellana, O., Cooley, L., and S?ll, D. (1986) Mol. Cell. Biol. 6, 525-529). We have examined the importance of the unique acceptor stem structure of E. coli tRNA(His) in determining the specificity of RNase P cleavage. Mutant tRNA(His) precursors bearing substitutions of the normal base G-1 or the opposing, potentially paired base, C73, can be cleaved at the +1 position, in contrast to wild-type precursors which are cut exclusively at the -1 position. These data indicate that the nature of the base at position -1 is of greater importance in determining the site of RNase P cleavage than potential base pairing between nucleotides -1 and 73. In addition, processing of the mutant precursors by M1-RNA or P RNA under conditions of ribozyme catalysis yields a higher proportion of +1-cleaved products in comparison to the reaction catalyzed by the RNase P holoenzyme. This lower sensitivity of the holoenzyme to alterations in acceptor stem structure suggests that the protein moiety of RNase P may play a role in determining the specificity of the reaction and implies that recognition of the substrate involves additional regions of the tRNA. We have also shown that the RNase P holoenzyme and tRNA(His) precursor of Saccharomyces cerevisiae, unlike their prokaryotic counterparts, do not possess these abilities to carry out this unusual reaction.     

RNase P activity in the mitochondria of Saccharomyces cerevisiae depends on both mitochondrion and nucleus-encoded components.

A requisite step in the biosynthesis of tRNA is the removal of 5' leader sequences from tRNA precursors. We have detected an RNase P activity in yeast mitochondrial extracts that can carry out this reaction on a homologous precursor tRNA. This mitochondrial RNase P was sensitive to both micrococcal nuclease and protease, demonstrating that it requires both a nucleic acid and protein for activity. The presence of RNase P activity in vitro directly correlated with the presence of a locus on yeast mitochondrial DNA previously shown by genetic and biochemical studies to be required for tRNA maturation. The product of the locus, the 9S RNA, and this newly described mitochondrial RNase P activity cofractionated, providing further evidence that the 9S RNA is the RNA component of yeast mitochondrial RNase P.     

Localization of human RNase Z isoforms: dual nuclear/mitochondrial targeting of the ELAC2 gene product by alternative translation initiation

RNase Z is an endonuclease responsible for the removal of 3' extensions from tRNA precursors, an essential step in tRNA biogenesis. Human cells contain a long form (RNase Z(L)) encoded by ELAC2, and a short form (RNase Z(S); ELAC1). We studied their subcellular localization by expression of proteins fused to green fluorescent protein. RNase Z(S) was found in the cytosol, whereas RNase Z(L) localized to the nucleus and mitochondria. We show that alternative translation initiation is responsible for the dual targeting of RNase Z(L). Due to the unfavorable context of the first AUG of ELAC2, translation apparently also starts from the second AUG, whereby the mitochondrial targeting sequence is lost and the protein is instead routed to the nucleus. Our data suggest that RNase Z(L) is the enzyme involved in both, nuclear and mitochondrial tRNA 3' end maturation.     

Nonsense suppression in Schizosaccharomyces pombe: the S. pombe Sup3-e tRNASerUGA gene is active in S. cerevisiae

Summary The gene encoding the efficient UGA suppressor sup3-e of Schizosaccharomyces pombe was isolated by in vivo transformation of Saccharomyces cerevisiae UGA mutants with S. pombe sup3-e DNA. DNA from a clone bank of EcoRI fragments from a S. pombe sup3-e strain in the hybrid yeast vector YRp17 was used to transform the S. cerevisiae multiple auxotroph his4-260 leu2-2 trp1-1 to prototrophy. Transformants were isolated at a low frequency; they lost the ability to grow in minimal medium after passaging in non-selective media. This suggested the presence of the suppressor gene on the non-integrative plasmid. Plasmid DNA, isolated from the transformed S. cerevisiae cells and subsequently amplified in E. coli, transformed S. cerevisiae his4-260 leu2-2 trp1-1 to prototrophy. In this way a 2.4 kb S. pombe DNA fragment carrying the sup3-e gene was isolated. Sequence analysis revealed the presence of two tRNA coding regions separated by a spacer of only seven nucleotides. The sup3-e tRNA _Ser ^UGA tRNA gene is followed by a sequence coding for the initiator tRNA^Met. The transformation results demonstrate that the cloned S. pombe UGA suppressor is active in S. cerevisiae UGA mutant strains.     

Purification and some properties of a specific nuclease which cleaves transfer RNA precursors from the posterior silk gland of Bombyx mori.

A specific endonuclease involved in the processing of tRNA precursors was isolated and partially purified from the posterior silk gland of Bombyx mori, and designated as RNase P.Bmo. This enzyme was shown to catalyze the conversion of 4.5 S precursor RNA to 4.1 S RNA by trimming the 5'-additional segment from the precursor RNA. RNase P.Bmo required divalent cations, Mg2+ or Mn2+. In the presence of these divalent cations, K+ or NH4+ activated the RNase P.Bmo reaction. Optimum pH was observed around 8.0. Ribosomal RNA's and mature tRNA from the silk gland were not cleaved by RNase P.Bmo. A 4.5 S precursor RNA fraction containing formycin, an adenosine analog, was less susceptible to RNase P.Bmo than the normal one. These results indicate that RNase P.Bmo has a high substrate specificity. An additional nuclease(s) was isolated. This activity was assumed to remove the extra 3'-segment of the 4.5 S precursor RNA.     

Metal ion and substrate structure dependence of the processing of tRNA precursors by RNase P and M1 RNA

A synthetic tRNA precursor analog containing the structural elements of Escherichia coli tRNA(Phe) was characterized as a substrate for E. coli ribonuclease P and for M1 RNA, the catalytic RNA subunit. Processing of the synthetic precursor exhibited a Mg2+ dependence quite similar to that of natural tRNA precursors such as E. coli tRNA(Tyr) precursor. It was found that Sr2+, Ca2+, and Ba2+ ions promoted processing of the dimeric precursor at Mg2+ concentrations otherwise insufficient to support processing; very similar behavior was noted for E. coli tRNA(Tyr). As noted previously for natural tRNA precursors, the absence of the 3'-terminal CA sequence in the synthetic precursor diminished the facility of processing of this substrate by RNase P and M1 RNA. A study of the Mg2+ dependence of processing of the synthetic tRNA dimeric substrate radiolabeled between C75 and A76 provided unequivocal evidence for an alteration in the actual site of processing by E. coli RNase P as a function of Mg2+ concentration. This property was subsequently demonstrated to obtain (Carter, B. J., Vold, B.S., and Hecht, S. M. (1990) J. Biol. Chem. 265, 7100-7103) for a mutant Bacillus subtilis tRNAHis precursor containing a potential A-C base pair at the end of the acceptor stem.     

Differential effects of mutations in the protein and RNA moieties of RNase P on the efficiency of suppression by various tRNA suppressors

We have studied the efficiency of suppression by tRNA suppressors in vivo in strains of Escherichia coli that harbor a mutation in the rnpA gene, the gene for the protein component (C5) of RNase P, and in strains that carry several different alleles of the rnpB gene, the gene for the RNA component (M1) of RNase P. Depending on the genetic background, different efficiencies of suppression by the various tRNA suppressors were observed. Thus, mutations in rnpA have separable and distinct effects from mutations in rnpB on the processing of tRNA precursors by RNase P. In addition, the efficiency of suppression by several derivatives of E. coli tRNA(Tyr) Su3 changed as the genetic background was altered.     

The RNase P ofDictostyelium discoideum

Ribonuclease P (RNase P) is a key enzyme involved in tRNA biosynthesis. It catalyses the endonucleolytic cleavage of nearly all tRNA precursors to produce 5-end matured tRNA. RNase P activity has been found in all organisms examined, from bacteria to mammals. Eubacterial RNase P RNA is the only known RNA enzyme which functionsin trans in nature. Similar behaviour has not been demonstrated in RNase P enzymes examined from archaebacteria or eukaryotes. Characterisation of RNase P enzymes from more diverse eukaryotic species, including the slime moldDictyostelium discoideum, is useful for comparative analysis of the structure and function of eukaryotic RNase P.Abbreviations RNase P ribonuclease P - MN micrococcal nuclease