RNase P RNA mediated cleavage: Substrate recognition and catalysis

Biochimie 89 (2007) 1183e1194 www.elsevier.com/locate/biochi

RNase P RNA mediated cleavage: Substrate recognition and catalysis Leif A. Kirsebom* Department of Cell and Molecular Biology, Box 596, Biomedical Centre, SE-751 24 Uppsala, Sweden Received 4 April 2007; accepted 25 May 2007 Available online 2 June 2007

Abstract The universally conserved endoribonuclease P consists of one RNA subunit and, depending on its origin, a variable number of protein subunits. RNase P is involved in the processing of a large variety of substrates in the cell, the preferred substrate being tRNA precursors. Cleavage activity does not require the presence of the protein subunit(s) in vitro. This is true for both prokaryotic and eukaryotic RNase P RNA suggesting that the RNA based catalytic activity has been preserved during evolution. Progress has been made in our understanding of the contribution of residues and chemical groups both in the substrate as well as in RNase P RNA to substrate binding and catalysis. Moreover, we have access to two crystal structures of bacterial RNase P RNA but we still lack the structure of RNase P RNA in complex with its substrate and/or the protein subunit. Nevertheless, these recent advancements put us in a new position to study the way and nature of interactions between in particular RNase P RNA and its substrate. In this review I will discuss various aspects of the RNA component of RNase P with an emphasis on our current understanding of the interaction between RNase P RNA and its substrate. Ó 2007 Elsevier Masson SAS. All rights reserved. Keywords: RNase P; Ribozyme; Divalent metal ions; tRNA precursors; tRNA processing

1. Introduction RNA research has undergone a revolution and it is now obvious that RNA molecules play widely diverse and fundamental roles in all living organisms. Apart from acting just as a messenger between chromosomal DNA and cellular proteins the functions that RNA carry out or participate in include RNA processing and protein translation, acting as structural scaffolds, transporters, gene regulators and biocatalysts. In fact most likely RNA, and not protein, constitutes the active centre where peptide bond formation takes place within the ribosome. However, the first RNA molecules that were demonstrated to act as biocatalysts were the Group I intron derived from Tetrahymena [1] and the RNA subunit of RNase P [2]. Since then several catalytic RNAs or ribozymes have been

* Tel.: þ46 18 471 4068; fax: þ46 18 53 03 96. E-mail address: [email protected] 0300-9084/$ - see front matter Ó 2007 Elsevier Masson SAS. All rights reserved. doi:10.1016/j.biochi.2007.05.009

identified: the hammerhead RNA, the hairpin and the Group II intron, to name a few [3]. The biological role of these as well as the Group I intron is to operate in cis, i.e. substrate and catalyst are located in the same RNA molecule. This is in contrast to RNase P RNA, which operates in trans and was the first trans acting ribozyme to be identified. RNase P RNA is part of a ribonucleoprotein complex involved in the processing of tRNA in both prokaryotic and eukaryotic cells. Based on biochemical and genetic data using bacterial RNase P we have good knowledge about which residues in RNase P RNA and in the substrate contribute to binding, cleavage site recognition and subsequent catalysis. In this review I will discuss our current understanding with an emphasis on the RNA component of RNase P. First I will discuss the composition of RNase P from various sources and this will be followed by discussions that relate to how the RNA recognizes its RNA substrate and the contribution of chemical groups in the substrate to catalysis. It is unavoidable to discuss RNA function without discussing the role of divalent metal

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ions, in particular Mg2þ, therefore I will also cover different aspects of the role Mg2þ in RNase P RNA mediated cleavage of RNA. 2. Biological role of RNase P and its composition The tRNA genes are transcribed as precursors and these need to be processed to give functional tRNA molecules. The endoribonuclease RNase P is responsible for generating tRNA with matured 50 ends and all tRNAs, with a few exceptions [4,5], carry a monophosphate at their 50 termini as a result of RNase P cleavage. Irrespective of origin, RNase P consists of one RNA and a variable number of protein subunits: one in Bacteria, four in Archaea, nine in yeast and ten in mammalian (human, nuclear) RNase P. The function and role of the RNase P protein subunits is beyond the scope of this review but it is known from studies of bacterial RNase P that the protein binds to the 50 leader of tRNA precursors and increases the binding affinity of the substrate [6,7]. In addition, the bacterial RNase P protein has been suggested to play a role in product release, prevention of rebinding of the substrate, the cleavage site

recognition process, broadening of substrate specificity and in stabilization of the native structure of RNase P RNA [8e14] (see also below). The protein is not needed for RNase P RNA to cleave its substrate in vitro but it is essential in vivo [15]. In this context it was demonstrated that the conserved human RNase P protein Rpp29 binds to Escherichia coli RNase P RNA, M1 RNA, resulting in a catalytically active complex [16,17]. However, as has recently been demonstrated, eukaryotic RNase P RNA has retained the basic capacity for substrate cleavage in the absence of its protein subunits [18] (see below). For a more detailed discussion about the function of the protein subunits in RNase P derived from Archaea and Eukarya I refer to two recent reviews by Gopalan and Altman [19] and Walker and Engelke [20]. 3. Catalytic activity resides in the RNA moiety Phylogenetic comparative analysis of RNase P RNA suggests that irrespective of origin the RNA can be folded into a common overall secondary structure with a conserved core (Fig. 1). Moreover, RNase P RNA can be divided into two

Fig. 1. Illustrations of the secondary structures of different types of RNase P RNA according to Haas and Brown [23]. The left structure represents the paradigm of type A, E. coli RNase P RNA, also termed M1 RNA. The type A M1 RNA is also shown in a schematic form together with type B (Mycoplasma hyopneumoniae), type M (Archaeoglobus fulgidus) and Eukarya (human) RNase P RNAs. The shaded areas represent the P7eP11 region in the specificity domain (here referred to as the TBS region) that interacts with the T-stem-loop region (TSL) of the precursor tRNA (referred to as the TSL/TBS interaction), residue A248 (A248/N1 interaction), the region in helix P4 suggested to interact with a 20 OH in the acceptor stem of the substrate [128] and the GGU-motif (highlighted in white) that interacts with the 30 end of the substrate (RCCAeRNase P RNA interaction). Note that the type M (A. fulgidus) and human RNase P RNA lack a GGU-motif (i.e. P15). The solid line in the left structure (between helices P5 and P7) marks the boarder between the specificity and catalytic domains. For details see main text.

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major domains, the specificity and the catalytic domains. The former carries the binding site (the P7eP11 region; Fig. 1) for the T-stem-loop region (TSL-region) of the tRNA precursor substrate [21,22] (Fig. 2, see also below) while the latter is involved in catalysis. Based on secondary structures (Fig. 1) bacterial RNase P RNA can be divided into two main types, type A (Ancestral type) and type B (Bacillus type). However, there are exceptions, e.g., Chlamydia spp. RNase P RNA that lacks the bacterial P15 loop structure (see below). Type A is also represented in various archaeal species together with type M. For RNase P RNA derived from eukaryotes there are some important structural differences that are relevant to what I will discuss below. First; the P15 loop region is missing and second; the A-rich bulge between P10 and P11 is absent in the specificity domain. Residues in the P15 loop pair with the

Fig. 2. The three dimensional structures of type A RNase P RNA from Thermatoga maritima [28] (top) and a precursor tRNA substrate (bottom) based on the structure of yeast tRNAPhe. The 50 leader of the precursor tRNA is indicated in red while the circles indicated with dashed lines represent the TBSregion (1), the region around residue U69 (M1 RNA numbering, see Fig. 1) that has been suggested to interact with a 20 OH in the acceptor stem (4) [130] and the region where residue A248 is located (3). The P15-loop with the GGU-motif (see Fig. 1) is not resolved in the structure (2). The dashed red circle indicates the T-stem, the acceptor stem, the 30 CCA-end and the 50 leader. The TSL refer to the T-stem-loop region of the tRNA precursor. The solid line indicates the distance (12 base pairs) from the T-loop to the RNase P cleavage site. For further details see the main text.

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30 end of the substrate while the A-rich bulge is part of the region that interacts with the characteristic TSL-region of tRNA precursor substrates as discussed above (see also below). For a more detailed discussion about phylogenetic comparative analysis of the RNA component of RNase P I refer to Haas and Brown [23], Frank et al. [24], Harris et al. [25], Marquez et al. [26] and Evans et al. [27]. Recently the crystal structures of two bacterial RNase P RNAs were solved, Thermatoga maritima [28] and Bacillus stearothermophilus [29] RNase P RNA. The former is a representative of a type A while the latter is of type B [23]. The secondary structures of the S-domains of type A and type B RNase P RNA are different. However, in spite of this the crystal structures of the S-domains of Thermus thermophilus and Bacillus subtilis revealed that the structure of this functionally important region is conserved [30]. The structure of the P15 loop on the other hand is less defined in both the full-size structures [28,29]. However, the NMR structure of a model of the M1 RNA P15 loop is available [31] and biochemical data suggest that the fold of the P15 loop is similar irrespective of whether it is part of the full-length RNase P RNA or part of a model RNA molecule. From biochemical data it is also apparent that the P15-loop is an autonomous metal ion-binding domain [32]. For further discussion in relation to the three dimensional structure of RNase P RNA I refer to Kazantsev and Pace [33]. Until 1999 only bacterial RNase P RNA (type A or B) had been demonstrated to mediate correct cleavage in the absence of protein. However, Brown and coworkers showed 1999 that RNase P RNA from some Archaea indeed are catalytically active at high salt concentrations [34]. This was only demonstrated to be the case for archaeal type A RNase P RNA and so far RNase P RNA of type M has not been shown to be catalytic in the absence of protein. We recently showed that both human and Giardia lamblia RNase P RNA mediate cleavage alone however at rates that are 106- to 107-fold lower compared to cleavage by E. coli RNase P RNA, the paradigm of type A RNase P RNA [18]. Thus, the capacity for RNA-based catalytic activity of RNase P has been preserved during evolution. The reason for the low activity observed for eukaryotic RNase P RNA alone needs to be clarified but it might be related, at least partly, to folding of the RNA and/or structural differences. As discussed above there are indeed significant structural differences between eukaryotic and bacterial RNase P RNA: e.g., the absence of the P15 loop and the A-rich bulge between P10 and P11. Nonetheless modeling of the three dimensional structure of eukaryotic RNase P RNA based on photoaffinity cross linking studies suggest that the eukaryal and bacterial RNase P RNA have a similar core structure [35]. Moreover, note that the type M RNase P RNA, found in Archaea, lacking the A-rich bulge between P10 and P11 has not been demonstrated to be active in the absence of proteins ([36] and references therein). Second, RNase P RNA derived from Chlamydia spp. that lack the P15-loop show very low cleavage activity without protein [23,37]. These observations are consistent with biochemical data demonstrating the

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importance of these regions for substrate binding and catalysis (see below). For further discussion I refer to refs. [19,35,36]. 4. RNase P RNA activity depends on divalent metal ions Understanding of RNA function is tightly linked to the role of divalent metal ions that are bound to the RNA, in particular Mg2þ (see, e.g., [38]). In the case of RNase P RNA approximately 100 Mg2þ ions bind to the RNA [39] and among these some are of particular importance in promoting correct folding of the RNA, facilitating the interaction with its RNA substrates and participating in the chemistry of cleavage. However, other metal ions also bind to the RNA; for example Ca2þ, which binds to RNA with approximately the same affinity as Mg2þ. Like Mg2þ, Ca2þ is of biological importance and it is abundant in the cell. Moreover, RNA cleavage activity changes as a function of the ratio between Ca2þ and Mg2þ as exemplified by the RNase P RNA system. This raises the possibility that RNA-based activities in the cell are up- or down-regulated depending on the intracellular concentrations of Mg2þ and Ca2þ[40]. In this section I will only discuss a few introductory aspects of RNase P RNA and Mg2þ; other more specific aspects will be discussed below. As in other large RNA molecules individual folding domains of RNase P RNA have been identified (e.g., [41,42]). The folding of RNase P RNA depends on Mg2þ and is suggested to be a cooperative process that is completed at 5e 10 mM [43e47]. Interestingly, lead(II) cleavage analysis of RNase P RNA in vivo and in vitro suggested that the overall conformation of the RNA in complex with the protein is not significantly altered by the intracellular environment [48]. Lead(II) induced cleavage of RNase P RNA in the presence of different divalent metal ions indicated that the RNA acquires a similar overall conformation in the presence of different metal ions, e.g., Mg2þ, Ca2þ, Mn2þ and Sr2þ while Pb2þ alone gives a different fold [49]. However, Pb2þ and Sr2þ cannot by themselves promote RNase P RNA-mediated cleavage at physiological pH while Pb2þ and Sr2þ together can [40,50,51]. This, together with other data, suggest at least two categories of metal ion in RNase P RNA mediate cleavage; one that promotes folding and the other is involved in generating the nucleophile. This is corroborated by the finding that RNase P RNA cleaves its substrate at the correct position even in the presence of Pb2þ and Co(NH3)3þ 6 [50]. It should also be mentioned that in the cell the folding of the RNA is related to the transcription process. However, this is beyond the scope of this review and has recently been discussed in another review by Pan and Sosnick [52]. With respect to catalysis, the available biochemical data suggest that the ( pro)-Rp oxygen at the cleavage site act as an inner ligand for Mg2þ coordinated at the cleavage site. The data indicate that this is the case for both bacterial RNase P RNA and eukaryotic RNase P holoenzymes [53e57]. Moreover, introduction of a single Sp-phosphorothioate modification at the cleavage site also affects catalysis. However, here it is less clear whether (pro)-Sp oxygen coordinates Mg2þ since the phenotype observed in the presence of the single

Sp-phosphorothioate modification cannot be rescued by replacing Mg2þ with thiophilic metal ions, Mn2þ or Cd2þ[53,55,56]. Finally, for other large ribozymes, e.g., Group I intron and ai5g Group II ribozymes, Mg2þ coordinates the 30 -oxyanion in the transition state [58e60]. However, as of today there is no experimental evidence indicating that this is the case in RNase P catalyzed reactions as well [61]. 5. Substrate interactions and RNase P The tRNA precursors within the cell are the preferred substrates for RNase P. In addition, there are a number of other RNA precursors that are processed by RNase P, e.g., the precursors to 4.5S RNA, tmRNA, bacteriophage M3 RNA and phage-derived antisense C4 RNA [9,62e65], mRNA [66] and E. coli RNA derived from non-coding intergenic regions and transient structures in riboswitches [67,68]. Several viral non-tRNAs such as TYMV RNA have been demonstrated to act as substrates in vitro [69]. In addition, several model substrates have been designed, some of which are cleaved with almost the same efficiency as regular tRNA precursor substrates [18,22,32,40,50,70e81]. In fact, we have recently shown that small precursors with 3 base pairs long stems act as substrates for RNase P RNA in the absence of the RNase P protein C5 [79]. In addition, it has been demonstrated that the bacterial RNase P holoenzyme cleaves small single stranded RNA [12,82]. This is in keeping with previous data demonstrating M1 RNA cleavage in single stranded regions both in the presence and in the absence of protein [83,84]. Based on these and other data a model rationalizing how RNase P recognizes and interacts with such a large number of different substrates is emerging (see below). Structural characterization of tRNA precursors suggested early that the tRNA domain has already acquired its characteristic fold at the precursor stage [85e89]. This is in keeping with defective RNase P processing caused by substitutions disrupting the tRNA structure. This suggested that RNase P recognizes the tRNA part of a precursor [90] and several experiments have confirmed this ([91] and references therein). However, it has been demonstrated that the protein (C5) of bacterial RNase P interacts with residues in the 50 leader of the precursor [92]. Following this, recent data from Harris and coworkers suggested that the interaction between C5 and the 50 leader serve to compensate for weaker interactions with the tRNA part. Thus, it appears that one role of C5 is to offset differences in tRNA precursor structures resulting in uniform binding and catalysis [7]. A productive interaction between the tRNA structure and RNase P RNA (Figs. 2 and 3B) has been demonstrated to include: (i) interaction between the TSL-region in the tRNA precursor and the P7eP11 region in the specificity domain (see above and Figs. 1 and 2; below I will refer to this region in the specificity domain as the TSL-binding site or TBS) (see also [22]); (ii) the RCCAeRNase P RNA interaction ([93], interacting residues in italic) where the RCC-motif at the 30 end of the tRNA precursor pairs with a conserved GGU-motif in the P15-loop region in RNase P RNA (Figs. 1 and 3B);

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Fig. 3. The secondary structures of two tRNA precursors (A) and models illustrating (B) the interactions between RNase P RNA and its substrate near the cleavage site and (C) detailed view of our current understanding of catalytically important functional groups and interactions at the site of cleavage. (A) Schematic illustration of the secondary structure of the precursors to tRNATyrSu3 and tRNAHis. CS indicates the cleavage sites and cleavage by bacterial RNase P generates 7 and 8 base pair long acceptor-stems as indicated (for details see the main text). The numbering 1 and þ1 at the cleavage site is to indicate that this is the canonical RNase P cleavage site. (B) The RCCAeRNase P RNA and A248/N1 interactions where interacting residues are highlighted in grey. The residues 2, 1,þ1,þ72 and þ76 near the cleavage site are as indicated. The exocyclic amine of Gþ1 and the 20 OH at 1 in the substrate have been demonstrated to contribute both to binding and catalysis (see the text). In the model MgA-MgC represents Mg2þ and A is suggested to stabilize the RCCAeRNase P RNA interaction while B is involved in generating the nucleophile. The arrow marks the canonical cleavage site. For historical reasons the interaction between the 30 end of the substrate is referred to as the RCCAeRNase P RNA interaction however based on our present understanding of this interaction R ¼ A, G and U. Consequently in the figure R ¼ N. C. The RNase P cleavage site where only one Mg2þ is shown; however available data clearly indicate that more than one Mg2þ ion is positioned in the vicinity of the cleavage site (see, e.g., [78] and references therein). Therefore, the shown Mg2þ ion represents more than one Mg2þ ion in the vicinity of the cleavage site. Consequently the indicated contacts (dashed lines) do not necessarily occur with the same Mg2þ ion. The chemical groups at the cleavage site that have been demonstrated to contribute to catalysis and/or binding are highlighted in grey. In addition N7 of Gþ73 have been suggested to play a role (although small) in cleavage site recognition. The question marks (?) refer to two alternatives: (i) the exocyclic amine (2NH2) at position 2 of Gþ1 can act as an outer sphere ligand for one of the Mg2þ positioned at and near the cleavage site or (ii) the 2NH2 is involved in an interaction with RNase P RNA that influences the positioning of Mg2þ in the vicinity of the cleavage site. At present we cannot distinguish between these two possibilities. aec represent chemical groups at the cleavage site that have been suggested to act as ligands (outer or inner sphere) for Mg2þ (for details see text). The arrow marks the phosphorus atom usually attacked in RNase P-mediated cleavage. The interaction between residue 248 in M1 RNA and the residue at 1 corresponds to the A248/N1 interaction while the interaction between 294 in M1 RNA and the residue at þ73 corresponds to the ‘‘þ73/294 interaction’’ that is part of the RCCAeRNase P RNA interaction (for detail and references see the main text).

(iii) the A248/N1 interaction where the residue immediately 50 of the scissile bond interacts with residue A248 in RNase P RNA [94]. We do not yet have access to crystal structures of RNase P RNA in complex with its substrate and therefore the nature of these interactions rely on genetic and biochemical data.

5.1. The TSL-/TBS interaction For the TSL-/TBS interaction it was inferred that the 20 OH of residues 54 and 56 as well as the exocyclic amine of C57 in the T-loop interact with groups in the TBS-region upon RNase P RNA substrate complex formation [95]. However, given that

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the TBS-region is also a region that binds Me2þ [51,96e100] involvement of metal ion(s) for the establishment of the TSL-/ TBS interaction can not be excluded. In the case of B. subtilis RNase P RNA it has also been suggested that the residue that corresponds to A233 in E. coli RNase P RNA interacts with the 20 OH at position 64 in the T-stem of a precursor to tRNAPhe [101]. Based on available data on the B. subtilis RNase P RNA system it was suggested that the TSL-region is a major determinant for specific binding of a precursor tRNA [95]. Our recent data using E. coli RNase P RNA are in keeping with this but we also provide evidence that the TSL-/TBS interaction results in a conformational change that influences the actual rate of cleavage [79]. Hence, this finding is consistent with an induced fit mechanism as discussed by GuerrierTakada et al. [102].

Genetic data for the RCCAeRNase P RNA interaction suggested that the bases C74 and C75 in the substrate base pair with the two G residues in the GGU-motif in the P15-loop [93,103] (Figs. 1 and 3B]. This finding is in keeping with chemical footprinting as well as other biochemical data (e.g., [104e108]). Such data also suggested that the C75/G292 pair is part of a triple base pair that involves A258 in the P15-loop [108,109] (Figs. 1 and 3B). Moreover, nucleotide analogue interference modification (NAIM) studies suggested that the Hoogsteen surface of A76 of the tRNA precursor interacts with G259 in RNase P RNA [108]. Recent data have also provided evidence that the RCCAeRNase P RNA interaction plays an important role in vivo [110,111]. Based on the RCCAeRNase P RNA interaction we proposed that this interaction anchors the substrate on the RNA, exposes the cleavage site and subsequently results in re-coordination of Mg2þ. This would ensure correct and efficient cleavage [93] (Fig. 3B). More recent data indicate the nature of the interaction between residues þ73 in the substrate and 294 in RNase P RNA (the þ73/294 interaction and E. coli numbering; Fig. 3C), which is an essential part of the RCCAeRNase P RNA interaction, plays an important role [50,76e78]. More specifically, both the identity and the orientation of this interaction, as well as to some extent the 20 OH and N7 of Gþ73 (when present) in the substrate contributes to catalysis, likely by affecting Mg2þ positioned in its vicinity and close to the cleavage site. Thus, we argued that there is cross talk between the þ73/294 interaction and the site of cleavage and that metal ion(s) take part in this process [50,78].

However, introducing a 3-methyl-U at 1 resulted in no apparent change in the affinity and in the rate of cleavage compared to having a U at this position. Hence, the A248/N1 interaction most likely does not involve the N3 position of U1 but it is not excluded that O2 and O4 are used [75]. Due to the presence of A, G and C at the 1 position in tRNA precursors the A248/N1 interaction must also accommodate A, G and C and in these cases cis WatsoneCrick/WatsoneCrick is excluded. Cross-linking data with 4-thioU at 1 and NAIM studies suggest that several residues in RNase P RNA are positioned close to the 1 residue: A248, A249, C252, C253, G332 and A333. This raises the possibility that these residues constitute a binding pocket/surface for the residue at 1 with A248 as a key residue. Then, dependent on the identity of the 1 residue different chemical groups in this pocket/ surface would interact with specific groups on the 1 residue [113e117]. This is consistent with the fact that cleavage is affected by the identity of the residue at the 1 position as demonstrated using different precursor substrates [75e77,79,94,118]. To resolve this we need the crystal structure of RNase P RNA in complex with its substrate. The identity of the residue at the 1 position influences either the affinity of the substrate or the rate of cleavage or both. However, this is dependent on the precursor, for example substituting the 1 residue in some tRNA precursors affect only binding and not the rate [94] while in the case of other precursors (tRNA precursors and model RNA substrates) both the rate and affinity are affected [76,77,79,80,119]. These findings emphasize the importance of using different appropriate substrates in order to elucidate the contribution of various residues/chemical groups in the substrate to catalysis. Moreover, upon RNase P RNA substrate complex formation the A248/N1 interaction most likely operates in conjunction with the RCCAeRNase P RNA interaction to ensure correct positioning for efficient cleavage of the scissile bonding. This is clearly evident in the cases where the 1 residue forms a strong pair with þ73 in the substrate, i.e., a C1/Gþ73 pair (see, e.g., [77,79] and references therein). For model RNA substrates as well as for tRNA precursors the data showed effects on both ground state binding and rate of cleavage due to substituting U1 with C1 (with G at þ73) such that substrates with C1 bound with less affinity and were cleaved with lower rates under single and multiple turnover conditions [77,80]. There is also a different Mg2þ requirement, for U vs C at the 1 position indicating the importance of Mg2þ in establishment of the A248/N1 interaction and/or for the opening of the N1/Nþ73 pair (when present) in the substrate.

5.3. The A248/N1 interaction

5.4. Importance of the P4 helix

Regarding the A248/N1 interaction it is less clear how this interaction is manifested. Genetic and biochemical data suggest that the residue at position 1 (residue immediately upstream of the RNase P cleavage site) interacts with the conserved A248 in RNase P RNA [94]. The most common nucleotide at 1 is U [77,94] and U can potentially establish a cis WatsoneCrick/WatsoneCrick base pair with A [94,112].

Phylogenetic comparative analysis revealed the presence of the P4 helix [120]. NAIM studies of tRNA binding as well as using a M1 RNA-substrate conjugate indicated the importance of residues in P4 for binding of functionally important metal ions [99,121e123]. Binding of metal ions in P4 is corroborated by structural studies of a small model RNA representing the P4 helix and by metal ion induced cleavage of full size

5.2. The RCCAeRNase P RNA interaction

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RNase P RNA [124e127]. However, metal ion binding does not appear to result in significant structural changes but might affect substrate binding. Fierke and coworkers [127] showed that specific phosphorothioate substitutions in P4 in the type B B. subtilis RNase P RNA lowered the rate of cleavage dramatically while binding was not affected. Base substitution/ deletion within P4 further emphasizes the importance of this region with respect to catalysis and metal ion binding. Comparing cleavage by wild type and mutant M1 RNA in which C70 had been replaced with U (or deletion of U69) showed an increased rate of cleavage for the mutants in the presence of Ca2þ as the only metal ion [123,129]. Together these data clearly point to the importance of P4 for both binding of Mg2þ and the substrate and subsequent cleavage. Recent cross-linking studies suggested that residues at and in the vicinity of U69 interact with the residue positioned five residues 30 of the cleavage site [130] (Fig. 2). As a consequence of this interaction the authors suggested that the metal binding site in P4 has a distal effect on catalytic metal ion(s) at the cleavage site.

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acts as an outer (or inner) sphere ligand for Mg2þ [78,134]. However, this interpretation has been questioned [112]. Nonetheless, to ensure correct cleavage products (with 50 phosphate and 30 OH) the strategy of RNase P RNA has to be to prevent the 20 OH at the cleavage site to act as a nucleophile (activated for example by Mg2þ) and favor a nucleophilic attack on the phosphorus from the other side. To accomplish this it is conceivable that the Mg2þ that coordinates the 20 OH also uses the 30 bridging oxygen as a ligand. For example this arrangement has been observed in the crystal structure of a catalytically active Group I intron intermediate [135] and biochemical data suggested that this is also the case in the transition state in Group II ribozyme cleavage [136]. However, attempts to demonstrate that the 30 -oxyanion coordinates Mg2þ in the transition state in RNase P RNA mediated cleavage has so far failed. But this does not exclude that this is indeed the case since these trials have been based on replacing the bridging oxygen with sulfur and this might result in exclusion of Mg2þ at this position [61]. To resolve this we have to wait until we have access to the crystal structure of RNase P RNA in complex with its substrate.

5.5. The role of the 20 OH at the cleavage site RNase P cleavage result in products with 50 phosphate and 3 OH while the adjacent 20 OH at the cleavage site remains unchanged. The 20 OH at the site of cleavage has been demonstrated to play an essential role in, e.g., metal ion induced cleavage of RNA and cleavage mediated by the hammerhead ribozyme where it acts as the nucleophile (see, e.g., [38]). In these cases products with 50 OH and 20 ;30 cyclic phosphates at their ends are generated. Focus has also been on the function of this 20 hydroxyl in RNase P cleavage: although it is not essential for cleavage its absence lowers the cleavage rate substantially. This was first demonstrated using nuclear RNase P derived from Saccharomyces cerevisiae and M1 RNA ([71] and references therein). This has been confirmed in several different laboratories using both full-size tRNA precursors and model substrates [72,73,75,78,95,112,119,131e134]. These studies show that replacement of the 20 OH at the cleavage site with 20 H or 20 NH2 results in changes in ground state binding, rate of cleavage, cleavage site recognition and metal ion binding in its vicinity. Moreover, it has been suggested that the 20 OH at the canonical cleavage site mediates an interaction between RNase P RNA and its substrate [119]. This model has been referred to as the ‘‘20 OH-model’’ and it predicts that disruption of this interaction would affect cleavage at other positions, e.g., at the position between residues 2 and 1. Although it cannot be excluded that the 20 OH at the cleavage site interacts with RNase P RNA, we and others have provided data that are inconsistent with the 20 OH-model [75,112]. These latter studies demonstrated that efficient cleavage depends on the 20 OH immediately 50 of the scissile bond irrespective of cleavage site and type of RNase P RNA (type A or B). Based on data using substrates with various modifications (20 H, 20 NH2 and 20 F) as well as cleavage at different pH and various metal ions it was suggested that in the RNase P RNA substrate complex the 20 hydroxyl at the cleavage site 0

5.6. The importance of having a G immediately 30 of the cleavage site The majority of tRNAs irrespective of origin carry a guanosine at the 50 termini [137]. It is well documented that Gþ1 (and the Gþ1/Cþ72 base pair) plays an important role in RNase P processing both in vivo and in vitro. Deletion or replacing Gþ1/Cþ72 affects cleavage site recognition, substrate binding and cleavage under physiological pH conditions as well as under conditions where chemistry of cleavage is suggested to be rate limiting [80,83,84,119,138e143]. In fact it has been suggested that the G at the þ1 position in a tRNA precursor acts as a guiding nucleotide [72,83]. Data based on a study of RNase P RNA-mediated cleavage of various precursors in which Gþ1/Cþ72 had been replaced with Uþ1/Aþ72 suggested that the identity of the þ1/þ72 pair is involved in substrate discrimination [80]. Moreover, cleavage of different model RNA substrate carrying modified residues at þ1 revealed that the exocyclic amine of Gþ1 contributes to substrate binding and catalysis as well as charge distribution and possibly Mg2þ binding at the cleavage site. Based on this we proposed that the exocyclic amine interacts with RNase P RNA and/or influences the positioning of Mg2þ in the vicinity of the cleavage site [81]. Interestingly, the exocyclic amine of Gþ1 at the cleavage site is positioned close to the 20 OH and N7 of Gþ73 that interacts with U294 in RNase P RNA, the þ73/294 interaction (Fig. 3C and not shown). The 20 OH and N7 of Gþ73 and the identity of the residues constituting the þ73/ 294 interaction appear to influence the positioning of metal ions in its vicinity and have been suggested to stabilize this interaction [77,78]. Hence, it is conceivable that the exocyclic amine of Gþ1 is involved in positioning of this metal ion perhaps by acting as an outer sphere ligand (metal ion A in Fig. 3B).

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5.7. Residue at the 2 position Monitoring cleavage efficiency of different 20 -modified tRNA precursor and model RNA substrates for a type A RNase P RNA revealed that the 20 OH at the 2 position in the 50 leader also plays a role in cleavage [72,130]. Loria and Pan used a type B RNase P RNA and their data suggested that substitution of the 20 OH at 2 with 20 H affects binding but not the rate of cleavage [119]. Two nucleotides long 50 leader vs a leader with only one residue increases the substrate affinity and rate of cleavage significantly [6]. The identity of the residue at this position in a tRNA precursor affects both cleavage site recognition and rate of cleavage [144]. However, the latter study also showed differences in the cleavage pattern comparing cleavage using type A and type B RNase P RNA that depended on the identity of the residue at 2. In the case of processing by RNase P of the E. coli tRNAfMet precursor that carries Cþ1 and Aþ72 (the majority of tRNA precursors have Gþ1/Cþ72), in vivo and in vitro studies indicated that G at 2 compensated for the lack of Gþ1 [145,146]. Together these data clearly suggest that the residue at the 2 position plays an important role in RNase P mediated cleavage. Moreover, it was suggested that this 20 OH is part of a functionally important Mg2þ binding site in the substrate [72] and that the true substrate for RNase P RNA is a substrate with Mg2þ bound in the vicinity of the cleavage site at the junction between single- and double-stranded regions [73].

5.8. Substrate and cleavage site recognition: bacterial vs eukaryotic RNase P RNA Bacterial RNase P processing of the tRNAHis precursor results in a 50 matured tRNA with an 8 base pair long amino acid acceptor-stem in contrast to most tRNAs that have 7 base pairs in their acceptor stems (Fig. 3A). In eukaryotes, on the other hand, RNase P cleave the precursor to tRNAHis generating an acceptor-stem with only 7 base pairs and the extra G is added by a specific enzyme after processing by RNase P [147,148]. This is consistent with a measuring mechanism in cleavage site recognition for the eukaryotic RNase P, marked with a solid line in Fig. 2 [149]. Moreover, it is well documented that bacterial RNase P, as well as its RNA, can cleave a number of different substrates including RNA hairpin model substrates that consist of a 50 leader, a helix representing the acceptor- and T-stems and a 30 CCA motif (see above). By contrast, the human RNase P holoenzyme did not cleave such RNA hairpin model substrates under standard assay conditions. Cleavage of this type of substrate required the presence of a bulged nucleotide positioned 7 nucleotides downstream of the cleavage site [150]. However, as its bacterial homologue eukaryotic RNase P RNA can, although with low efficiency, cleave a RNA hairpin model substrate with no bulged nucleotide. In addition, it appears that bacterial and human RNase P RNA cleave a bacterial tRNAHis precursor at the same position generating an 8 base pair long acceptor stem [18] (see also Fig. 3A). These observations

suggest that cleavage and cleavage site recognition have been preserved in the RNA during the evolution. Hence, this raises the intriguing possibility that the proteins in eukaryotic RNase P indeed have a role in specifying the cleavage site and perhaps also in narrowing substrate specificity. In the case of cleavage of the bacterial tRNAHis precursor we cannot exclude the possibility of inefficient cleavage between two G residues, i.e., between G1 and Gþ1 (and/or the presence of a G1/Cþ72 base pair in the substrate; Fig. 3A) in the human RNase P RNA alone reaction [75,138e140,151]. Neither can it be excluded that the reason for the observed differences might be that Kikovska et al. [18] used different reaction conditions compared to previous studies. Nonetheless, an important future question will be to understand the role of the different protein subunits in eukaryotic RNase P in the cleavage recognition process and with respect to substrate specificity.

6. Concluding remarks The preferred substrate for RNase P is precursor tRNA, however, several other RNA molecules in the cell are also processed by RNase P (see above). Several determinants (see Fig. 2) in the substrate have been identified and reducing the number of determinants in general results in less efficient processing. For example, comparing RNase P RNA mediated cleavage of substrate variants with Gþ1/Cþ72 and Uþ1/Aþ72 provided evidence for substrate discrimination both at the level of binding and at the catalytic step [80]. However, as recent data suggest, structural elements in one region of the precursor can compensate for a less optimal structure in another region [7]. Together, the data demonstrates complexity in orchestration of the various determinants that result in efficient cleavage and emphasize the importance of the substrate in RNase P mediated cleavage. Moreover, given that various substrates are processed with different efficiencies suggests a role for RNase P in the regulation of gene expression. From biochemical data we have information about contact points between RNase P RNA and its substrate but there is a need for structural information based on high-resolution structures of RNase P RNA in complex with its substrate. Also, solving the structure of the RNase P holoenzyme with and without the substrate will reveal valuable and critical information to get a more detailed picture of the nature of the interactions between RNase P and its different substrates as well as of the subsequent cleavage process. Finally, that the catalytic activity of RNase P has been demonstrated to reside in the RNA also for eukaryotic RNase P opens up new possibilities to study and determine the role of its various protein subunits. Moreover, we are now in the position to identify residues in eukaryotic RNase P RNA that interact with specific nucleotides in the substrate leading to a productive RNase P RNA substrate interaction. Obviously these are only some of the questions that need to be answered to understand this central and conserved ribonucleoprotein in more detail.

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Acknowledgements I thank my colleagues with whom I have been working with over the years. I also acknowledge Drs S. Das Gupta and S.G. Sva¨rd for discussions and critical reading of the manuscript. The ongoing work in my laboratory is funded by the Swedish Research Council (to L.A.K. and to Uppsala RNA Research Center in the form of Linnesto¨d) and by the Foundation for strategic research.

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