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.     

Sequence changes in both flanking sequences of a pre-tRNA influence the cleavage specificity of RNase P.

The cleavage specificities of the RNase P holoenzymes from Escherichia coli and the yeast Schizosaccharomyces pombe and of the catalytic M1 RNA from E. coli were analyzed in 5'-processing experiments using a yeast serine pre-tRNA with mutations in both flanking sequences. The template DNAs were obtained by enzymatic reactions in vitro and transcribed with phage SP6 or T7 RNA polymerase. The various mutations did not alter the cleavage specificity of the yeast RNase P holoenzyme; cleavage always occurred predominantly at position G + 1, generating the typical seven base-pair acceptor stem. In contrast, the specificity of the prokaryotic RNase P activities, i.e. the catalytic M1 RNA and the RNase P holoenzyme from E. coli, was influenced by some of the mutated pre-tRNA substrates, which resulted in an unusual cleavage pattern, generating extended acceptor stems. The bases G - 1 and C + 73, forming the eighth base pair in these extended acceptor stems, were an important motif in promoting the unusual cleavage pattern. It was found only in some natural pre-tRNAs, including tRNA(SeCys) from E. coli, and tRNAs(His) from bacteria and chloroplasts. Also, the corresponding mature tRNAs in vivo contain an eight base pair acceptor stem. The presence of the CCA sequence at the 3' end of the tRNA moiety is known to enhance the cleavage efficiency with the catalytic M1 RNA. Surprisingly, the presence or absence of this sequence in two of our substrate mutants drastically altered the cleavage specificity of M1 RNA and of the E. coli holoenzyme, respectively. Possible reasons for the different cleavage specificities of the enzymes, the influence of sequence alterations and the importance of stacking forces in the acceptor stems are discussed.     

Minimum requirements for substrates of mammalian tRNA 3' processing endoribonuclease.

Mammalian tRNA 3' processing endoribonuclease (3' tRNase) removes a 3' trailer after the discriminator nucleotide from precursor tRNA (pre-tRNA). To elucidate the minimum requirements for 3' tRNase substrates, we tested small pre-tRNA(Arg) substrates lacking the D and anticodon stem-loop domain for cleavage by purified pig 3' tRNase. A small pre-tRNA (R-ATW) composed of an acceptor stem, an extra loop, a T stem-loop domain, a discriminator nucleotide, and a 3' trailer was cleaved more efficiently than the full-length wild type. The catalytic efficiencies of three R-ATW derivatives, which were constructed to destroy the original T stem base pairs, were also higher than that of the full-length wild type. Pig 3' tRNase efficiently processed a "minihelix" (R-ATM5) that consists of a T stem-loop domain, an acceptor stem, a discriminator nucleotide, and a 3' trailer, while the enzyme never cleaved a "microhelix" that is composed of a T loop, an acceptor stem, a discriminator nucleotide, and a 3' trailer. Five R-ATM5 derivatives that have one to seven base substitutions in the T loop were all cleaved slightly more efficiently than the full-length wild type and slightly less efficiently than R-ATM5. A helix ("minihelixDelta1") one base pair smaller than minihelices was a good substrate, while small helices containing a continuous 10-base pair stem were poor substrates. The cleavage of these three small substrates occurred after the discriminator and one to three nucleotides downstream of the discriminator. From these results, we conclude that minimum substrates for efficient cleavage by mammalian 3' tRNase are minihelices or minihelicesDelta1, in which there seem to be no essential bases.     

Recognition of the 5' leader of pre-tRNA substrates by the active site of ribonuclease P

The bacterial tRNA processing enzyme ribonuclease P (RNase P) is a ribonucleoprotein composed of a approximately 400 nucleotide RNA and a smaller protein subunit. It has been established that RNase P RNA contacts the mature tRNA portion of pre-tRNA substrates, whereas RNase P protein interacts with the 5' leader sequence. However, specific interactions with substrate nucleotides flanking the cleavage site have not previously been defined. Here we provide evidence for an interaction between a conserved adenosine, A248 in the Escherichia coli ribozyme, and N(-1), the substrate nucleotide immediately 5' of the cleavage site. Specifically, mutations at A248 result in miscleavage of substrates containing a 2' deoxy modification at N(-1). Compensatory mutations at N(-1) restore correct cleavage in both the RNA-alone and holoenzyme reactions, and also rescue defects in binding thermodynamics caused by A248 mutation. Analysis of pre-tRNA leader sequences in Bacteria and Archaea reveals a conserved preference for U at N(-1), suggesting that an interaction between A248 and N(-1) is common among RNase P enzymes. These results provide the first direct evidence for RNase P RNA interactions with the substrate cleavage site, and show that RNA and protein cooperate in leader sequence recognition.     

Effects of 5' leader and 3' trailer structures on pre-tRNA processing by nuclear RNase P

Eukaryotic transfer RNA precursors (pre-tRNAs) contain a 5' leader preceding the aminoacyl acceptor stem and a 3' trailer extending beyond this stem. An early step in pre-tRNA maturation is removal of the 5' leader by the endoribonuclease, RNase P. Extensive pairing between leader and trailer sequences has previously been demonstrated to block RNase P cleavage, suggesting that the 5' leader and 3' trailer sequences might need to be separated for the substrate to be recognized by the eukaryotic holoenzyme. To address whether the nuclear RNase P holoenzyme recognizes the 5' leader and 3' trailer sequences independently, interactions of RNase P with pre-tRNA(Tyr) containing either the 5' leader, the 3' trailer, or both were examined. Kinetic analysis revealed little effect of the 3' trailer or a long 5' leader on the catalytic rate (k(cat)) for cleavage using the various pre-tRNA derivatives. However, the presence of a 3' trailer that pairs with the 5' leader increases the K(m) of pre-tRNA slightly, in agreement with previous results. Similarly, competition studies demonstrate that removal of a complementary 3' trailer lowers the apparent K(I), consistent with the structure between these two sequences interfering with their interaction with the enzyme. Deletion of both the 5' and 3' extensions to give mature termini resulted in the least effective competitor. Further studies showed that the nuclear holoenzyme, but not the B. subtilis holoenzyme, had a high affinity for single-stranded RNA in the absence of attached tRNA structure. The data suggest that yeast nuclear RNase P contains a minimum of two binding sites involved in substrate recognition, one that interacts with tRNA and one that interacts with the 3' trailer. Furthermore, base pairing between the 5' leader and 3' trailer hinders recognition.     

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.     

Control of the position of RNase P-mediated transfer RNA precursor processing

Two Bacillus subtilis tRNA(His) precursors (Green, C. J., and Vold, B. S. (1988) J. Biol. Chem. 263, 652-657) were processed by Escherichia coli RNase P in the presence of varying [Mg2+]. The wild type precursor was processed under all conditions to afford a single tRNA product containing 8 base pairs in the acceptor stem. In contrast, the position of processing of a mutant tRNA(His) precursor (containing a G27----A27 alteration) was shown to be condition-dependent. Processing occurred at A27 under conditions consistent with formation of an A27-C100 base pair in the acceptor stem but at G28 under conditions that disfavored base pair formation. The ability to control the site of RNase P-mediated tRNA precursor processing is unprecedented and permits analysis of the chemical factors that promote processing.     

Cooperative RNP assembly: complementary rescue of structural defects by protein and RNA subunits of archaeal RNase P

Ribonuclease P (RNase P) is a ribonucleoprotein complex that utilizes a Mg(2+)-dependent RNA catalyst to cleave the 5' leader of precursor tRNAs (pre-tRNAs) and generate mature tRNAs. The bacterial RNase P protein (RPP) aids RNase P RNA (RPR) catalysis by promoting substrate binding, Mg(2+) coordination and product release. Archaeal RNase P comprises an RPR and at least four RPPs, which have eukaryal homologs and function as two binary complexes (POP5·RPP30 and RPP21·RPP29). Here, we employed a previously characterized substrate-enzyme conjugate [pre-tRNA(Tyr)-Methanocaldococcus jannaschii (Mja) RPR] to investigate the functional role of a universally conserved uridine in a bulge-helix structure in archaeal RPRs. Deletion of this bulged uridine resulted in an 80-fold decrease in the self-cleavage rate of pre-tRNA(Tyr)-MjaΔU RPR compared to the wild type, and this defect was partially ameliorated upon addition of either RPP pair. The catalytic defect in the archaeal mutant RPR mirrors that reported in a bacterial RPR and highlights a parallel in their active sites. Furthermore, an N-terminal deletion mutant of Pyrococcus furiosus (Pfu) RPP29 that is defective in assembling with its binary partner RPP21, as assessed by isothermal titration calorimetry and NMR spectroscopy, is functional when reconstituted with the cognate Pfu RPR. Collectively, these results indicate that archaeal RPPs are able to compensate for structural defects in their cognate RPR and vice-versa, and provide striking examples of the cooperative subunit interactions critical for driving archaeal RNase P toward its functional conformation.     

Splicing of a yeast proline tRNA containing a novel suppressor mutation in the anticodon stem

The intron-containing proline tRNAUGG genes in Saccharomyces cerevisiae can mutate to suppress +1 frameshift mutations in proline codons via a G to U base substitution mutation at position 39. The mutation alters the 3' splice junction and disrupts the bottom base-pair of the anticodon stem which presumably allows the tRNA to read a four-base codon. In order to understand the mechanism of suppression and to study the splicing of suppressor pre-tRNA, we determined the sequences of the mature wild-type and mutant suppressor gene products in vivo and analyzed splicing of the corresponding pre-tRNAs in vitro. We show that a novel tRNA isolated from suppressor strains is the product of frameshift suppressor genes. Sequence analysis indicated that suppressor pre-tRNA is spliced at the same sites as wild-type pre-tRNA. The tRNA therefore contains a four-base anticodon stem and nine-base anticodon loop. Analysis of suppressor pre-tRNA in vitro revealed that endonuclease cleavage at the 3' splice junction occurred with reduced efficiency compared to wild-type. In addition, reduced accumulation of mature suppressor tRNA was observed in a combined cleavage and ligation reaction. These results suggest that cleavage at the 3' splice junction is inefficient but not abolished. The novel tRNA from suppressor strains was shown to be the functional agent of suppression by deleting the intron from a suppressor gene. The tRNA produced in vivo from this gene is identical to that of the product of an intron+ gene, indicating that the intron is not required for proper base modification. The product of the intron- gene is a more efficient suppressor than the product of an intron+ gene. One interpretation of this result is that inefficient splicing in vivo may be limiting the steady-state level of mature suppressor tRNA.     

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.     

The kinetics and specificity of cleavage by RNase P is mainly dependent on the structure of the amino acid acceptor stem.

Cleavage by RNase P of the tRNA(His precursor yields a mature tRNA with an 8 base pair amino acid acceptor stem instead of the usual 7 base pair stem. Here we show, both in vivo and in vitro, that this is mainly dependent on the primary structure and length of the acceptor stem in the precursor. Furthermore, the tRNA(His) precursor used in this study was processed with a change in both kinetic constants, Km and kcat, in comparison to the kinetics of cleavage of the precursor to tRNA(Tyr)Su3. Cleavage of a chimeric tRNA precursor showed that these altered kinetics were due to a difference in the primary structure and in the length of the acceptor stems of these two tRNA precursors. We also studied the cleavage reaction as a function of base substitutions at positions -1 and/or +73 in the precursor to tRNA(His). Our results suggest that the nucleotide at position +73 in tRNA(His) plays a significant role in the kinetics of cleavage of its precursor, possibly in product release. In addition, it appears that the C5 protein of RNase P is involved in the interaction between the enzyme and its substrate in a substrate-dependent manner, as previously suggested.     

The exocyclic amine at the RNase P cleavage site contributes to substrate binding and catalysis

Most tRNAs carry a G at their 5' termini, i.e. at position +1. This position corresponds to the position immediately downstream of the site of cleavage in tRNA precursors. Here we studied RNase P RNA-mediated cleavage of substrates carrying substitutions/modifications at position +1 in the absence of the RNase P protein, C5, to investigate the role of G at the RNase P cleavage site. We present data suggesting that the exocyclic amine (2NH2) of G+1 contributes to cleavage site recognition, ground state binding and catalysis by affecting the rate of cleavage. This is in contrast to O6, N7 and 2'OH that are suggested to affect ground state binding and rate of cleavage to significantly lesser extent. We also provide evidence that the effects caused by the absence of 2NH2 at position +1 influenced the charge distribution and conceivably Mg2+ binding at the RNase P cleavage site. These findings are consistent with models where the 2NH2 at the cleavage site (when present) interacts with RNase P RNA and/or influences the positioning of Mg2+ in the vicinity of the cleavage site. Moreover, our data suggest that the presence of the base at +1 is not essential for cleavage but its presence suppresses miscleavage and dramatically increases the rate of cleavage. Together our findings provide reasons why most tRNAs carry a guanosine at their 5' end.     

Differences in the interaction of Escherichia coli RNase P RNA with tRNAs containing a short or a long extra arm.

The phosphorothioate footprinting technique was applied to the investigation of phosphate moieties in tRNA substrates involved in interactions with M1 RNA, the catalytic subunit of Escherichia coli RNase P. In general agreement with previous data, all affected sites were localized in acceptor stem and T arm. But the analyzed examples for class I (Saccharomyces cerevisiae pre-tRNA(Phe) with short variable arm) and class II tRNAs (E. coli pre-tRNA(Tyr) with large variable arm) revealed substantial differences. In the complex with pre-tRNA(Phe), protection was observed at U55, C56, and G57, along the top of the T loop in the tertiary structure, whereas in pre-tRNA(Tyr), the protected positions were G57, A58, and A59, at the bottom of the T loop. These differences suggest that the size of the variable arm affects the spatial arrangement of the T arm, providing a possible explanation for the discrepancy in reports about the D arm requirement in truncated tRNA substrates for eukaryotic RNase P enzymes. Enhanced reactivities were found near the junction of acceptor and T stem (U6, 7, 8 in pre-tRNA(Phe) and G7, U63, U64 in pre-tRNA(Tyr)). This indicates a partial unfolding of the tRNA structure upon complex formation with RNase P RNA.     

Nucleotides in precursor tRNAs that are required intact for catalysis by RNase P RNAs.

Precursor tRNAAsp molecules, containing a 26-base 5' leader, were treated with diethylpyrocarbonate, 50% hydrazine or anhydrous hydrazine/3M NaCl and then subjected to processing by RNase P RNAs from Escherichia coli or Bacillus subtilis. Fully processed tRNAs and material not successfully cleaved by the catalytic RNAs were analyzed for their content of chemically altered nucleotides. Several bases were identified as being required intact for optimal activity as substrate as judged by exclusion of chemically modified residues from processed molecules, and simultaneous enhancement in material that was not recognized as substrate. Such nucleotides cluster near the site of cleavage at the mature 5' end and in the T stem and loop. Purines at residues 1 and 2 adjacent to the site of cleavage, position 57 in the T loop, and site 64 in the T stem exhibited the most pronounced effects. These results suggest a model of recognition of substrate by RNase P RNAs in which the ribozyme interacts with the corner of the precursor tRNA's three dimensional structure, where the T- and D-loops are juxtaposed, and extends along the top of the molecule back towards the site of catalysis.     

Site selection by Xenopus laevis RNAase P

Investigation of the mechanism of cleavage site selection by Xenopus RNAase P reveals that the acceptor stem, a 7 bp helix common to all tRNA precursors, is required for cleavage. We propose that Xenopus RNAase P recognizes conserved features of the mature tRNA and that the cleavage site is selected by measuring the length of the acceptor stem. In support of this, we demonstrate that insertion of 2 bp in the acceptor stem of yeast pre-tRNA(3Leu) relocates the cleavage site 2 bases 3' to the original one. In addition, insertion of 1 bp in the acceptor stem of the end-matured yeast pre-tRNA(Phe) generates an RNAase P cleavage site: the enzyme produces a mature tRNA with the characteristic 7 bp stem and releases one 5' flanking nucleotide. Since it has previously been shown that cleavage sites of the splicing endonuclease are determined by the length of the anticodon stem, RNAase P and the splicing endonuclease apparently use different stems to determine their cutting sites.