Complexity in orchestration of chemical groups near different cleavage sites in RNase P RNA mediated cleavage

RNase P mediated cleavage of the tRNA(His) precursor does not rely on the formation of the "+73/294 interaction" to give the correct cleavage product, i.e. cleavage at -1, while other tRNA precursors that are cleaved at the canonical site +1 do. A previous model, here referred to as the "2'OH-model", predicts that the 2'OH at the canonical cleavage site would affect cleavage at -1. Here we used model RNA hairpin substrates mimicking the structural architecture of the tRNA(His) precursor cleavage site to investigate the role of 2'OH with respect to ground state binding and rate of cleavage in the presence and absence of the +73/294 interaction. Our data emphasize the importance of the 2'OH in the immediate vicinity of the scissile bond. Moreover, introduction of 2'H at the cleavage site did not affect cleavage at an alternative cleavage site to any significant extent. Our findings are therefore inconsistent with the 2'OH model. We favor a model where the 2'OH at the cleavage site influence Mg2+ binding in its vicinity, however we do not exclude the possibility that the 2'OH at the cleavage site interacts with RNase P RNA. Studying the importance of the 2'OH at different cleavage sites also indicated a higher dependence on the 2'OH at the cleavage site in the absence of the +73/294 interaction than in its presence. Finally, we provide data suggesting that N3 of U at position -1 in the substrate is most likely not involved in an interaction with RNase P RNA.     

Base pairing between Escherichia coli RNase P RNA and its substrate.

Base pairing between the substrate and the ribozyme has previously been shown to be essential for catalytic activity of most ribozymes, but not for RNase P RNA. By using compensatory mutations we have demonstrated the importance of Watson-Crick complementarity between two well-conserved residues in Escherichia coli RNase P RNA (M1 RNA), G292 and G293, and two residues in the substrate, +74C and +75C (the first and second C residues in CCA). We suggest that these nucleotides base pair (G292/+75C and G293/+74C) in the ribozyme-substrate complex and as a consequence the amino acid acceptor stem of the precursor is partly unfolded. Thus, a function of M1 RNA is to anchor the substrate through this base pairing, thereby exposing the cleavage site such that cleavage is accomplished at the correct position. Our data also suggest possible base pairing between U294 in M1 RNA and the discriminator base at position +73 of the precursor. Our findings are also discussed in terms of evolution.     

Importance of the +73/294 interaction in Escherichia coli RNase P RNA substrate complexes for cleavage and metal ion coordination

We have studied an interaction, the "73/294-interaction", between residues 294 in M1 RNA (the catalytic subunit of Escherichia coli RNase P) and +73 in the tRNA precursor substrate. The 73/294-interaction is part of the "RCCA-RNase P RNA interaction", which anchors the 3' R(+73)CCA-motif of the substrate to M1 RNA (interacting residues underlined). Considering that in a large fraction of tRNA precursors residue +73 is base-paired to nucleotide -1 immediately 5' of the cleavage site, formation of the 73/294-interaction results in exposure of the cleavage site. We show that the nature/orientation of the 73/294-interaction is important for cleavage site recognition and cleavage efficiency. Our data further suggest that this interaction is part of a metal ion-binding site and that specific chemical groups are likely to act as ligands in binding of Mg(2+) or other divalent cations important for function. We argue that this Mg(2+) is involved in metal ion cooperativity in M1 RNA-mediated cleavage. Moreover, we suggest that the 73/294-interaction operates in concert with displacement of residue -1 in the substrate to ensure efficient and correct cleavage. The possibility that the residue at -1 binds to a specific binding surface/pocket in M1 RNA is discussed. Our data finally rationalize why the preferred residue at position 294 in M1 RNA is U.     

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.     

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.     

Evidence for induced fit in bacterial RNase P RNA-mediated cleavage

RNase P with its catalytic RNA subunit is involved in the processing of a number of RNA precursors with different structures. However, precursor tRNAs are the most abundant substrates for RNase P. Available data suggest that a tRNA is folded into its characteristic structure already at the precursor state and that RNase P recognizes this structure. The tRNA D-/T-loop domain (TSL-region) is suggested to interact with the specificity domain of RNase P RNA while residues in the catalytic domain interact with the cleavage site. Here, we have studied the consequences of a productive interaction between the TSL-region and its binding site (TBS) in the specificity domain using tRNA precursors and various hairpin-loop model substrates. The different substrates were analyzed with respect to cleavage site recognition, ground-state binding, cleavage as a function of the concentration of Mg(2+) and the rate of cleavage under conditions where chemistry is suggested to be rate limiting using wild-type Escherichia coli RNase P RNA, M1 RNA, and M1 RNA variants with structural changes in the TBS-region. On the basis of our data, we conclude that a productive TSL/TBS interaction results in a conformational change in the M1 RNA substrate complex that has an effect on catalysis. Moreover, it is likely that this conformational change comprises positioning of chemical groups (and Mg(2+)) at and in the vicinity of the cleavage site. Hence, our findings are consistent with an induced-fit mechanism in RNase P RNA-mediated cleavage.     

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.     

The recognition by RNase P of precursor tRNAs

We have generated mutants of M1 RNA, the catalytic subunit of Escherichia coli RNaseP, and have analyzed their properties in vitro and in vivo. The mutations, A333----C333, A334----U334, and A333 A334----C333 U334 are within the sequence UGAAU which is complementary to the GT psi CR sequence found in loop IV of all E. coli tRNAs. We have examined: 1) whether the mutant M1 RNAs are active in processing wild type tRNA precursors and 2) whether they can restore the processing defect in mutant tRNA precursors with changes within the GT psi CR sequence. As substrates for in vitro studies we used wild type E. coli SuIII tRNA(Tyr) precursor, and pTyrA54, a mutant tRNA precursor with a base change that could potentially complement the U334 mutation in M1 RNA. The C333 mutation had no effect on activity of M1 RNA on wild type pTyr. The U334 mutant M1 RNA, on the other hand, had a much lower activity on wild type pTyr. However, use of pTyrA54 as substrate instead of wild type pTyr did not restore the activity of the U334 mutant M1 RNA. These results suggest that interactions via base pairing between nucleotides 331-335 of M1 RNA and the GT psi CG of pTyr are probably not essential for cleavage of these tRNA precursors by M1 RNA. For assays of in vivo function, we examined the ability of mutant M1 RNAs to complement a ts mutation in the protein component of RNaseP in FS101, a recA- derivative of E. coli strain A49. In contrast to wild type M1 RNA, which complements the ts mutation when it is overproduced, neither the C333 nor the U334 mutant M1 RNAs was able to do so.     

Functional coupling between a distal interaction and the cleavage site in bacterial RNase-P-RNA-mediated cleavage

Bacterial RNase P consists of one protein and one RNA [RNase P RNA (RPR)]. RPR can process tRNA precursors correctly in the absence of the protein. Here we have used model hairpin loop substrates corresponding to the acceptor, T-stem, and T-loop of a precursor tRNA to study the importance of the T-loop structure in RPR-alone reaction. T-stem/loop (TSL) interacts with a region in RPR [TSL binding site (TBS)], forming TSL/TBS interaction. Altering the T-loop structure affects both cleavage site selection and rate of cleavage at the correct site + 1 and at the alternative site − 1. The magnitude of variation depended on the structures of the T-loop and the TBS region, with as much as a 150-fold reduction in the rate of cleavage at + 1. Interestingly, for one T-loop structure mutant, no difference in the rate at − 1 was detected compared to cleavage of the substrate with an unchanged T-loop, indicating that, in this case, the altered T-loop structure primarily influences events required for efficient cleavage at the correct site + 1. We also provide data supporting a functional link between a productive TSL/TBS interaction and events at the cleavage site. Collectively, our findings emphasize the interplay between separate regions upon formation of a productive RPR substrate that leads to efficient and accurate cleavage. These new data provide support for an induced-fit mechanism in bacterial RPR-mediated cleavage at the correct site + 1.     

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.     

Determinants of Escherichia coli RNase P cleavage site selection: a detailed in vitro and in vivo analysis.

The location of the Escherichia coli RNase P cleavage site was studied both in vitro and in vivo. We show that selection of the cleavage site is dependent on the nucleotide at the cleavage site and the length of the acceptor-stem. Within the acceptor-stem the number of nucleotides on the 5'-half of the acceptor-stem appears to be the important determinant, rather than the number of base pairs in the acceptor-stem. We also demonstrate that the length of the T-stem and a G to C substitution at position 57 in the tRNA(Tyr)Su3 precursor influence the location of the cleavage site under certain conditions. With respect to the function of the subunits of RNase P our data suggest that the nucleotide at position 333 in M1 RNA, and the C5 protein, are important for the identification of the cleavage site.     

Splice junction mutations in a yeast tRNA gene which alter the rate and precision of processing

We have introduced mutations into a tRNALeu3 gene which alter the intron boundaries and examined their effects on RNA splicing. Our results show that the 5'-proximal splice junction is not specified by the position of an adjacent base-paired stem present in all naturally occurring tRNA precursors. Also, efficient cleavage of 5'-splice junctions unique to these mutants, -CpU-, -UpA- and -UpG-, indicates the purine found at the 5'-side of this site in all natural precursors is dispensable. Some alterations of the sequence and structure at the 5'-proximal splice site reduce the rate of cleavage therein and result in accumulation of molecules composed of the 5'-half of the tRNA plus the intron. The precise position of the 5'-proximal cleavage site can vary +/- 1 base in these mutants. The 3'-proximal splice junction is rendered inactive by changing the prospective splice junction sequence from -ApC- to -CpC- and reducing the size of an unpaired loop at this site from six to two bases. Very small amounts of RNA composed of the 3'-half of the tRNA plus the intron accumulate from this precursor. We conclude that splice junction sequence and structure affect both the rate and precision of intervening sequence removal.     

Product release is a rate-limiting step during cleavage by the catalytic RNA subunit of Escherichia coli RNase P.

The kinetic constants for cleavage of the tRNA(Tyr)Su3 precursor by the M1 RNA of E. coli RNase P were determined in the absence and presence of the C5 protein under single and multiple (steady state) turnover conditions. The rate constant of cleavage in the reaction catalyzed by M1 RNA alone was 5 times higher in single turnover than in multiple turnovers, suggesting that a rate-limiting step is product release. Cleavage by M1 RNA alone and by the holoenzyme under identical buffer conditions demonstrated that C5 facilitated product release. Addition of different product-like molecules under single turnover reaction conditions inhibited cleavage both in the absence and presence of C5. In the presence of C5, the Ki value for matured tRNA was approximately 20 times higher than in its absence, suggesting that C5 also reduces the interaction between the 5'-matured tRNA and the enzyme. In a growing cell the number of tRNA molecules is approximately 1000 times higher than the number of RNase P molecules. A 100-fold excess of matured tRNA over enzyme clearly inhibited cleavage in vitro. We discuss the possibility that RNase P is involved in the regulation of tRNA expression under certain growth conditions.     

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.     

Several regions of a tRNA precursor determine the Escherichia coli RNase P cleavage site.

The RNase P cleavage reaction was studied as a function of the number of base-pairs in the acceptor-stem and/or T-stem of a natural tRNA precursor, the tRNA(Tyr)Su3 precursor. Our data suggest that the location of the Escherichia coli RNase P cleavage site does not depend merely on the lengths of the acceptor-stem and T-stem as previously suggested. Surprisingly, we find that precursors with only four base-pairs in the acceptor-stem are cleaved by M1 RNA and by holoenzyme. Furthermore, we show that both disruption of base-pairing, and alteration of the nucleotide sequence (without disruption of base-pairing) proximal to the cleavage site result in aberrant cleavage. Thus, the identity of the nucleotides near the cleavage site is important for recognition of the cleavage site rather than base-pairing. The important nucleotides are those at positions -2, -1, +1, +72, +73 and +74. We propose that the nucleotide at position +1 functions as a guiding nucleotide. These results raise the possibility that Mg2+ binding near the cleavage site is dependent on the identity of the nucleotides at these positions. In addition, we show that disruption of base-pairing in the acceptor-stem affects both Michaelis-Menten constants, Km and kcat.     

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.     

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.