Phylogenetic comparative mutational analysis of the base-pairing between RNase P RNA and its substrate.

We have studied the base-pairing between the 3'-terminal CCA motif of a tRNA precursor and RNase P RNA by a phylogenetic mutational comparative approach. Thus, various derivatives of the Escherichia coli tRNA(Ser)Su1 precursor harboring all possible substitutions at either the first or the second C of the 3'-terminal CCA motif were generated. Cleavage site selection on these precursors was studied using mutant variants of M1 RNA, the catalytic subunit of E. coli RNase P, carrying changes at positions 292 or 293, which are involved in the interaction with the 3'-terminal CCA motif. From our data we conclude that these two C's in the substrate interact with the well-conserved G292 and G293 through canonical Watson-Crick base-pairing. Cleavage performed using reconstituted holoenzyme complexes suggests that this interaction also occurs in the presence of the C5 protein. Furthermore, we studied the interaction using various derivatives of RNase P RNAs from Mycoplasma hyopneumoniae and Mycobacterium tuberculosis. Our results suggest that the base-pairing between the 3'-terminal CCA motif and RNase P is present also in other bacterial RNase P-substrate complexes and is not limited to a particular bacterial species.     

A novel endonucleolytic mechanism to generate the CCA 3' termini of tRNA molecules in Thermotoga maritima

The tRNA 3'-terminal CCA sequence is essential for aminoacylation of the tRNAs and for translation on the ribosome. The tRNAs are transcribed as larger precursor molecules containing 5' and 3' extra sequences. In the tRNAs that do not have the encoded CCA, the 3' extra sequence after the discriminator nucleotide is usually cleaved off by the tRNA 3' processing endoribonuclease (3' tRNase, or RNase Z), and the 3'-terminal CCA residues are added thereto. Here we analyzed Thermotoga maritima 3' tRNase for enzymatic properties using various pre-tRNAs from T. maritima, in which all 46 tRNA genes encode CCA with only one exception. We found that the enzyme has the unprecedented activity that cleaves CCA-containing pre-tRNAs precisely after the CCA sequence, not after the discriminator. The assays for pre-tRNA variants suggest that the CA residues at nucleotides 75 and 76 are required for the enzyme to cleave pre-tRNAs after A at nucleotide 76 and that the cleavage occurs after nucleotide 75 if the sequence is not CA. Intriguingly, the pre-tRNA(Met) that is the only T. maritima pre-tRNA without the encoded CCA was cleaved after the discriminator. The kinetics data imply the existence of a CCA binding domain in T. maritima 3' tRNase. We also identified two amino acid residues critical for the cleavage site selection and several residues essential for the catalysis. Analysis of cleavage sites by 3' tRNases from another eubacteria Escherichia coli and two archaea Thermoplasma acidophilum and Pyrobaculum aerophilum corroborates the importance of the two amino acid residues for the cleavage site selection.     

Protection from chemical modification of nucleotides in complexes of M1 RNA, the catalytic subunit of RNase P from E coli, and tRNA precursors.

Certain nucleotides in M1 RNA, the catalytic RNA subunit of RNase P from E coli, are protected from chemical modification when M1 RNA forms complexes with tRNA precursor molecules (ES complexes). Many of these nucleotides are important in the formation of the Michaelis complex. In the presence of tRNA precursor molecules, the pattern of protection from chemical modification of a region in M1 RNA that resembles the E site in 23S rRNA is similar to the pattern of protection of the E site in the presence of deacylated tRNA. In the complex with the RNA enzyme, more nucleotides in the substrate become accessible to modification, an indication that the substrate is in an unfolded conformation under these conditions.     

The nucleotide sequence of a precursor to the glycine- and threonine-specific transfer ribonucleic acids of Escherichia coli.

The primary nucleotide sequence of an Escherichia coli tRNA precursor molecule has been determined. This precursor RNA, specified by the transducing phage lambdah80dglyTsuA36 thrT tyrT, accumulates in a mutant strain temperature-sensitive for RNase P activity. The 170-nucleotide precursor RNA is processed by E. coli extracts to form mature tRNA Gly 2 suA36 and tRNA Thr ACU/C. The sequence of the precursor is pG-U-U-C-C-A-G-G-A-U-G-C-G-G-G-C-A-U-C-G-U-A-U-A-A-U-G-G-C-U-A-U-U-A-C-C-U-C-A-G-C-C-U-N-C-U-A-A-G-C-U-G-A-U-G-A-U-G-C-G-G-G-T-psi-C-G-A-U-U-C-C-C-G-C-U-G-C-C-C-G-C-U-C-C-A-A-G-A-U-G-U-G-C-U-G-A-U-A-U-A-G-C-U-C-A-G-D-D-G-G-D-A-G-A-G-C-G-C-A-C-C-C-U-U-G-G-U-mt6A-A-G-G-G-U-G-A-G-m7G-U-C-G-G-C-A-G-T-psi-C-G-A-A-U-C-U-G-C-C-U-A-U-C-A-G-C-A-C-C-A-C-U-UOH(tRNA sequences are italicized). It contains the entire primary nucleotide sequences of tRNA Gly2 suA36 and tRNA Thr ACU/C, including the common 3'-terminal sequence, CCA. Nineteen additional nucleotides are present, with 10 at the 5' end, 3 at the 3' end, and the remaining 6 in the inter-tRNA spacer region. RNase P cleaves the precursor specifically at the 5' ends of the mature tRNA sequences.     

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.     

The RNase P RNA from cyanobacteria: short tandemly repeated repetitive (STRR) sequences are present within the RNase P RNA gene in heterocyst-forming cyanobacteria.

The RNase P RNA gene (rnpB) from 10 cyanobacteria has been characterized. These new RNAs, together with the previously available ones, provide a comprehensive data set of RNase P RNA from diverse cyanobacterial lineages. All heterocystous cyanobacteria, but none of the non-heterocystous strains analyzed, contain short tandemly repeated repetitive (STRR) sequences that increase the length of helix P12. Site-directed mutagenesis experiments indicate that the STRR sequences are not required for catalytic activity in vitro. STRR sequences seem to have recently and independently invaded the RNase P RNA genes in heterocyst-forming cyanobacteria because closely related strains contain unrelated STRR sequences. Most cyanobacteria RNase P RNAs lack the sequence GGU in the loop connecting helices P15 and P16 that has been established to interact with the 3'-end CCA in precursor tRNA substrates in other bacteria. This character is shared with plastid RNase P RNA. Helix P6 is longer than usual in most cyanobacteria as well as in plastid RNase P RNA.     

Sequence-dependent cleavage site selection by RNase Z from the cyanobacterium Synechocystis sp. PCC 6803

Biosynthesis of transfer RNA requires processing from longer precursors at the 5'- and 3'-ends. In eukaryotes, in archaea, and in those bacteria where the 3'-terminal CCA sequence is not encoded, 3' processing is carried out by the endonuclease RNase Z, which cleaves after the discriminator nucleotide to generate a mature 3'-end ready for the addition of the CCA sequence. We have identified and cloned the gene coding for RNase Z in the cyanobacterium Synechocystis sp. PCC 6803. The gene has been expressed in Escherichia coli, and the recombinant protein was purified. The enzymatic activity of RNase Z from Synechocystis has been studied in vitro with a variety of substrates. The presence of C or CC after the discriminator nucleotide modifies the cleavage site of RNase Z so that it is displaced by one and two nucleotides to the 3'-side, respectively. The presence of the complete 3'-terminal CCA sequence in the precursor of the tRNA completely inhibits RNase Z activity. The inactive CCA-containing precursor binds to Synechocystis RNase Z with similar affinity than the mature tRNA. The properties of the enzyme described here could be related with the mechanism by which CCA is added in this organism, with the participation of two separate nucleotidyl transferases, one specific for the addition of C and another for the addition of A. This work is the first characterization of RNase Z from a cyanobacterium, and the first from an organism with two separate nucleotidyl transferases.     

RNase MRP and RNase P share a common substrate.

RNase MRP is a site-specific ribonucleoprotein endoribonuclease that processes RNA from the mammalian mitochondrial displacement loop containing region. RNase P is a site-specific ribonucleoprotein endoribonuclease that processes pre-tRNAs to generate their mature 5'-ends. A similar structure for the RNase P and RNase MRP RNAs and a common cleavage mechanism for RNase MRP and RNase P enzymes have been proposed. Experiments with protein synthesis antibiotics have shown that both RNase MRP and RNase P are inhibited by puromycin. We also show that E. coli RNase P cleaves the RNase MRP substrate, mouse mitochondrial primer RNA, exactly at a site that is cleaved by RNase MRP.     

Rp-phosphorothioate modifications in RNase P RNA that interfere with tRNA binding.

We have used Rp-phosphorothioate modifications and a binding interference assay to analyse the role of phosphate oxygens in tRNA recognition by Escherichia coli ribonuclease P (RNase P) RNA. Total (100%) Rp-phosphorothioate modification at A, C or G positions of RNase P RNA strongly impaired tRNA binding and pre-tRNA processing, while effects were less pronounced at U positions. Partially modified E. coli RNase P RNAs were separated into tRNA binding and non-binding fractions by gel retardation. Rp-phosphorothioate modifications that interfered with tRNA binding were found 5' of nucleotides A67, G68, U69, C70, C71, G72, A130, A132, A248, A249, G300, A317, A330, A352, C353 and C354. Manganese rescue at positions U69, C70, A130 and A132 identified, for the first time, sites of direct metal ion coordination in RNase P RNA. Most sites of interference are at strongly conserved nucleotides and nine reside within a long-range base-pairing interaction present in all known RNase P RNAs. In contrast to RNase P RNA, 100% Rp-phosphorothioate substitutions in tRNA showed only moderate effects on binding to RNase P RNAs from E. coli, Bacillus subtilis and Chromatium vinosum, suggesting that pro-Rp phosphate oxygens of mature tRNA contribute relatively little to the formation of the tRNA-RNase P RNA complex.     

Characterization of the RNase P RNA of Sulfolobus acidocaldarius.

RNase P is the ribonucleoprotein enzyme that cleaves precursor sequences from the 5' ends of pre-tRNAs. In Bacteria, the RNA subunit is the catalytic moiety. Eucaryal and archaeal RNase P activities copurify with RNAs, which have not been shown to be catalytic. We report here the analysis of the RNase P RNA from the thermoacidophilic archaeon Sulfolobus acidocaldarius. The holoenzyme was highly purified, and extracted RNA was used to identify the RNase P RNA gene. The nucleotide sequence of the gene was determined, and a secondary structure is proposed. The RNA was not observed to be catalytic by itself, but it nevertheless is similar in sequence and structure to bacterial RNase P RNA. The marked similarity of the RNase P RNA from S. acidocaldarius and that from Haloferax volcanii, the other known archael RNase P RNA, supports the coherence of Archaea as a phylogenetic domain.     

Processing of a multimeric tRNA precursor from Bacillus subtilis by the RNA component of RNase P

Processing of multimeric precursor tRNAs from Bacillus subtilis by the catalytic RNA component of RNase P was studied in vitro. Previous studies on processing by either Escherichia coli or B. subtilis RNase P-RNA utilized monomeric or dimeric substrates. In the experiments described here, a multimeric precursor tRNA containing six complete tRNA sequences and the partial sequence of a seventh were used. One species did not encode the 3'-terminal CCA sequence and the partial tRNA lacked 3' nucleotides and could form only a 3-base pair instead of a 7-base paired aminoacyl stem. Two species had the potential for forming extended base-paired aminoacyl stems. Processing was studied under varied ionic conditions. Chemical sequencing of the products showed that the RNase P-RNA cleavage produced the proper mature 5' termini for all of the six complete tRNA species, but no 5'-cleavage of the partial species was observed. At suboptimal ionic concentrations, the two species capable of forming extended base-paired aminoacyl stems were not observed. Thus, encoding of the 3'-CCA in a tRNA species is not critical for processing, but the formation of an aminoacyl stem with more than 3 base pairs is necessary. Particularly noteworthy was the observation that all species of the multimeric precursor could be processed at significantly lower ionic conditions than monomeric precursors used previously by ourselves and others. However, a single precursor species produced from the multimeric precursor could also be processed at the same lower ionic conditions as the multimeric precursor. This demonstrates that precursor tRNA species can differ widely in their ionic requirements for processing and that, to a large extent, the optimal conditions of MgCl2 or NH4Cl are a function of the substrate which is used.     

Recognition of RNA encapsidation signal by the yeast L-A double-stranded RNA virus

The encapsidation signal of the yeast L-A virus contains a 24-nucleotide stem-loop structure with a 5-nucleotide loop and an A bulged at the 5' side of the stem. The Pol part of the Gag-Pol fusion protein is responsible for encapsidation of viral RNA. Opened empty viral particles containing Gag-Pol specifically bind to this encapsidation signal in vitro. We found that binding to empty particles protected the bulged A and the flanking-two nucleotides from cleavage by Fe(II)-EDTA-generated hydroxyl radicals. The five nucleotides of the loop sequence ((4190)GAUCC(4194)) were not protected. However, T1 RNase protection and in vitro mutagenesis experiments indicated that G(4190) is essential for binding. Although the sequence of the other four nucleotides of the loop is not essential, data from RNase protection and chemical modification experiments suggested that C(4194) was also directly involved in binding to empty particles rather than indirectly through its potential base pairing with G(4190). These results suggest that the Pol domain of Gag-Pol contacts the encapsidation signal at two sites: one, the bulged A, and the other, G and C bases at the opening of the loop. These two sites are conserved in the encapsidation signal of M1, a satellite RNA of the L-A virus.     

In vitro processing of B. mori transfer RNA precursor molecules.

Ribonuclease P and 3'-5' nuclease, two enzymatic activities necessary for tRNA synthesis in E. coli, are also found in the silkgland cells of Bombyx mori. B. mori subcellular extracts containing RNAase P activity can cleave the E. coli tRNA precursor molecule endonucleolytically at the same site as the E. coli enzyme, and will also cleave in vitro all E. coli tRNA precursors (pre-tRNAs) which the bacterial enzyme recognizes. B. mori RNAase P will not cleave two E. coli RNAase P substrates that are structurally unrelated to tRNA. Pre-tRNAs from B. mori contain extra 5' and 3' nucleotides as judged by RNA fingerprinting and 5' terminal phosphate analysis. Crude silkgland extracts containing both RNAase P and 3'-5' nuclease can remove the 5' and 3' extra nucleotides from B. mori pre-tRNAs, whereas purified fractions containing RNAase P remove only 5' extra nucleotides. Only large silkworm pre-tRNAs were found to be susceptible to cleavage by B. mori RNAase P. This observation and sequence analysis of intermediates of in vitro processing reactions indicate a two-step process of pre-tRNA maturation in which extra 5' nucleotides are first removed by RNAase P and extra 3' nucleotides are then trimmed off by a 3'-5' nuclease.     

Identification of nucleotide residues essential for RNase P activity from the hyperthermophilic archaeon Pyrococcus horikoshii OT3

Ribonuclease P (RNase P) is involved in the processing of the 5' leader sequence of precursor tRNA (pre-tRNA). We have found that RNase P RNA (PhopRNA) and five proteins (PhoPop5, PhoRpp21, PhoRpp29, PhoRpp30, and PhoRpp38) reconstitute RNase P activity with enzymatic properties similar to those of the authentic ribozyme from the hyperthermophilic archaeon Pyrococcus horikoshii OT3. We report here that nucleotides A40, A41, and U44 at helix P4, and G269 and G270 located at L15/16 in PhopRNA, are, like the corresponding residues in Esherichia coli RNase P RNA (M1RNA), involved in hydrolysis by coordinating catalytic Mg(2+) ions, and in the recognition of the acceptor end (CCA) of pre-tRNA by base-pairing, respectively. The information reported here strongly suggests that PhopRNA catalyzes the hydrolysis of pre-tRNA in approximately the same manner as eubacterial RNase P RNAs, even though it has no enzymatic activity in the absence of the proteins.     

Structural requirements for processing of synthetic tRNAHis precursors by the catalytic RNA component of RNase P

Experiments were conducted to investigate structural features of the aminoacyl stem region of precursor histidine tRNA critical for the proper cleavage by the catalytic RNA component of RNase P that is responsible for 5' maturation. Histidine tRNA was chosen for study because tRNAHis has an 8 base pair instead of the typical 7-base pair aminoacyl stem. The importance of the 3' proximal CCA sequence in the 5'-processing reaction was also investigated. Our results show that the tRNAHis precursor patterned after the natural Bacillus subtilis gene is cleaved by catalytic RNAs from B. subtilis or Escherichia coli, leaving an extra G residue at the 5'-end of the aminoacyl stem. Replacing the 3' proximal CCA sequence in the substrate still allowed the catalytic RNA to cleave at the proper position, but it increased the Km of the reaction. Changing the sequence of the 3' leader region to increase the length of the aminoacyl stem did not alter the cleavage site but reduced the reaction rate. However, replacing the G residue at the expected 5' mature end by an A changed the processing site, resulting in the creation of a 7-base pair aminoacyl stem. The Km of this reaction was not substantially altered. These experiments indicate that the extra 5' G residue in B. subtilis tRNAHis is left on by RNase P processing because of the precursor's structure at the aminoacyl stem and that the cleavage site can be altered by a single base change. We have also shown that the catalytic RNA alone from either B. subtilis or E. coli is capable of cleaving a precursor tRNA in which the 3' proximal CCA sequence is replaced by other nucleotides.     

Pentatricopeptide repeats of protein-only RNase P use a distinct mode to recognize conserved bases and structural elements of pre-tRNA

Pentatricopeptide repeat (PPR) motifs are α-helical structures known for their modular recognition of single-stranded RNA sequences with each motif in a tandem array binding to a single nucleotide. Protein-only RNase P 1 (PRORP1) in Arabidopsis thaliana is an endoribonuclease that uses its PPR domain to recognize precursor tRNAs (pre-tRNAs) as it catalyzes removal of the 5′-leader sequence from pre-tRNAs with its NYN metallonuclease domain. To gain insight into the mechanism by which PRORP1 recognizes tRNA, we determined a crystal structure of the PPR domain in complex with yeast tRNA^Phe at 2.85 Å resolution. The PPR domain of PRORP1 bound to the structurally conserved elbow of tRNA and recognized conserved structural features of tRNAs using mechanisms that are different from the established single-stranded RNA recognition mode of PPR motifs. The PRORP1 PPR domain-tRNA^Phe structure revealed a conformational change of the PPR domain upon tRNA binding and moreover demonstrated the need for pronounced overall flexibility in the PRORP1 enzyme conformation for substrate recognition and catalysis. The PRORP1 PPR motifs have evolved strategies for protein-tRNA interaction analogous to tRNA recognition by the RNA component of ribonucleoprotein RNase P and other catalytic RNAs, indicating convergence on a common solution for tRNA substrate recognition.