The Exocyclic Amine at the RNase P Cleavage Site Contributes to Substrate Binding and Catalysis

doi:10.1016/j.jmb.2006.03.040

J. Mol. Biol. (2006) 359, 572–584

The Exocyclic Amine at the RNase P Cleavage Site Contributes to Substrate Binding and Catalysis Ema Kikovska, Mathias Bra¨nnvall and Leif A. Kirsebom* Department of Cell and Molecular Biology, Uppsala University, Box 596, Biomedical Centre, SE-751 24 Uppsala Sweden

Most tRNAs carry a G at their 5 0 termini, i.e. at position C1. 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 C1 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 GC1 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 0 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 C1 influenced the charge distribution and conceivably Mg2C 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 Mg2C in the vicinity of the cleavage site. Moreover, our data suggest that the presence of the base at C1 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 0 end. q 2006 Elsevier Ltd. All rights reserved.

*Corresponding author

Keywords: RNase P; ribozyme; divalent metal ions; tRNA precursors; RNA processing

Introduction The tRNA genes known to date are expressed as precursors and RNase P with its catalytic RNA subunit (RNase P RNA) is the endoribonuclease responsible for generating tRNA molecules with matured 5 0 ends. The residue at the 5 0 end is referred to as the C1 residue both in the matured tRNA and in the corresponding precursor. Available data suggest that RNase P recognizes the global tRNA structure and that the aminoacyl acceptor-stem is a major determinant.1 Recently we provided experimental evidence of substrate discrimination in RNase P RNA-mediated cleavage and that the C1/C72 basepair, i.e. the first base-pair in the aminoacyl acceptorstem of tRNA plays an essential role in this discrimination.2 The importance of the C1/C72 base-pair is consistent with earlier findings demonstrating that replacement of the guanosine at the C1 position (GC1) influences cleavage site recognition, the rate of cleavage (cleavage efficiency) and the level Abbreviation used: RS, RNase P RNA–substrate. E-mail address of the corresponding author: [email protected]

of matured tRNA in vivo.3–11 It was suggested that GC1 in the matured tRNA acts as a guiding nucleotide in the RNase P cleavage reaction.8,12 The importance of GC1 was further augmented by the finding that introduction of inosine at the cleavage site in a tRNAHis precursor as well as in a model tRNA precursor substrate affected cleavage site recognition.9,13 Moreover, guanosine has been demonstrated to play important mechanistic roles in RNA-mediated cleavage for a number of different ribozymes.14 To understand the function of guanosine in RNAmediated cleavage of RNA in further detail in particular with respect to RNase P RNA-promoted catalysis we decided to study the contribution of individual chemical groups of the guanosine (GC1) at the RNase P cleavage site to catalysis. This is of particular relevance given that most tRNAs in bacteria and eukaryotic cells carry a guanosine at the 5 0 termini.15 Here we present data suggesting that the exocyclic amine (2NH2) of GC1 contributes to cleavage site recognition, ground state binding and catalysis by affecting the rate of cleavage, demonstrating its importance in RNase P RNA-mediated catalysis.

0022-2836/$ - see front matter q 2006 Elsevier Ltd. All rights reserved.

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RNase P Cleavage and tRNA Precursors Processing

This is in contrast to O6, N7 and 2 0 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 the exocyclic amine at position C1 influenced the charge distribution and conceivably Mg2C binding at the RNase P cleavage site. Moreover, our data suggest that the presence of the base at C1 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 this position.

Results To study the role of different chemical groups of the guanosine at the RNase P cleavage site (i.e. at the C1 position) in RNase P RNA-mediated cleavage we decided to use the well-characterized model RNA hairpin substrate pATSer (Figure 1). RNase P RNA cleaves this substrate between positions C1 and K1 consequently position C1 corresponds to the first position of the 5 0 matured cleavage product. One advantage with this type of model substrate compared to tRNA precursors is that they can be synthesized chemically and therefore base modifications can easily be introduced at any position of choice. In addition, we have shown elsewhere that ground state binding and kinetics of cleavage using pATSer derivatives and regular tRNA precursors are comparable.2,16–21 Moreover, recent data suggest that the residue at K1 interacts with residue A248 in the RNase P RNA–substrate (RS) complex. This interaction is referred to as “the A248/NK1 interaction” (Figure 1 18,22). Therefore, substrates with substitutions/modifications at C1 both in UK1 and CK1 backgrounds were used. The residue that pairs with GC1 in pATSer corresponds to CC72 in tRNA and is referred to as NC72 throughout this study (Figure 1; see also Table 1). In accordance with this, the position immediately 3 0 of NC72 will be referred to as NC73. The different substrates listed in Table 1 were subjected to cleavage using M1 RNA (RNase P RNA derived from Escherichia coli) and we monitored: cleavage efficiency under multiple turnover conditions at physiological pH (kcat/Km), cleavage site recognition, cleavage as a function of [Mg2C], ground state binding (appKd) and rate of cleavage under saturating single turnover conditions at pH 6.0 where chemistry is suggested to be ratelimiting (kobs)2,20 (and references therein). All experiments were conducted in the absence of the RNase P protein C5 since available data suggest that the C5 protein does not interact with the residue at the C1 position23 (and references therein). Our previous21 as well as unpublished Pb2C-induced cleavage data (Table 1) suggest that the various modifications only influenced the local structural environment at the cleavage site and not the overall conformation of the substrate.

Impact of GC1 on the efficiency of cleavage under multiple turnover conditions Previously we reported the efficiency of cleavage for model RNA hairpin (pATSer) substrates as a function of changes at and near the RNase P cleavage site under multiple turnover conditions (for references see above). For comparison we therefore first determined the efficiency of cleavage of pATSer variants carrying base substitutions/modifications at positions C1 and/or C72 in the CK1 context (see Figure 1 and Table 1) under multiple turnover conditions at physiological pH (7.2) at 160 mM Mg2C. The data are summarized in Table 2. Importance of the 2NH2 of GC1 Replacing GC1 with any of the purin analogues tested resulted in decreased kcat/Km values, indicating the importance to have G at position C1. However, the reduction in cleavage was not as dramatic as in the case when GC1/CC72 had been replaced with UC1/AC72 (Table 2).2 Changing CK1 to UK1 and GC73 to AC73 resulted in higher kcat/Km values in keeping with our previous data,2,19 while the effect on the cleavage efficiency was less pronounced as a result of replacing GC1 with 2APC1 or AC1 in the UK1/AC73 context (Table 2 and data not shown). These results indicated the importance of GC1/CC72 in RNase P RNA-mediated cleavage and extend our previous findings where we studied cleavage as a function of replacing GC1/CC72 with UC1/AC72 (or UC1/DAPC72) in different substrate contexts.2 This conclusion is further supported by our studies where we studied cleavage under single turnover conditions at pH 7.2 of different tRNATyrSu3 precursor (pSu3) derivatives carrying base substitutions at C1 and/or C72 (Figure 2). Here we observed reductions in the rate of cleavage to varying degrees. But, in contrast to what we observed using the pATSer substrates we only detected cleavage at the canonical cleavage site, i.e. at C1 in the case of the pSu3 derivatives (data not shown; with respect to cleavage site recognition for the pATSer derivatives, see below). Importance of the 2 0 OH and N7 of GC1 Our multiple turnover data also revealed a reduction in kcat/Km as a result of replacing the 2 0 OH of GC1 with 2 0 H (Table 2; compare GC1/CC72 with dGC1/CC72; throughout this study d corresponds to 2 0 H while c7 is referred to substitution of N7 with carbon i.e. 7-deazaguanosine) whereas substitution N7 with c7 did not affect cleavage efficiency to any significant extent at least not in the context with a 2 0 H at C1 (Table 2; compare dGC1/ C C72 with c7dG C1/C C72). Thus, these data suggested that the 2 0 OH of GC1 plays a role in RNase P RNA-mediated cleavage at least in the CK1 context (see also below). This is in keeping with previous data using a similar type of model substrate.12

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RNase P Cleavage and tRNA Precursors Processing

Figure 1. (a) Secondary structures of the different model substrates pATSer used in the present study. The Y indicated in red corresponds to a pyrimidine (U or C) at these positions. The underlined residues correspond to the identities of residues at the C1 and C72 positions, i.e. the C1/C72 pair. Ino, inosine; 2AP, 2-amino purine; DAP, 2;6-diamino purine; Pu, purine; IsoG, iso-guanosine; DMA, N6, N6-dimethyl-adenosine; Rib, ribavirin; d refers to substitution of the 2 0 OH to 2 0 H; c7 refers to replacement of N7 to c7 of guanosine; D, deletion of the base, i.e. the guanosine. The black arrow and cs indicate the canonical RNase P cleavage site (between residues K1 and C1) while red Y (or U) denotes the residue at position K1. (b) Secondary structure of the precursor to tRNATyrSu3 (pSu3). The C1/C72 pair is highlighted in red and where substituted as indicated in Figure 2. The black arrow and cs denote the canonical RNase P cleavage site. (c) Illustration of the RNase P cleavage site and the residue at position C1, GC1. The chemical groups of GC1, the 2 0 OH at K1 and at C1 that were deleted/substituted are indicated, where DZdeletion.

Importance of the presence of the base at C1 To our surprise deleting the base (dDC1/CC72) at C1 still resulted in cleavage at C1 as well as at other positions (see below). But, the efficiency of cleavage was significantly reduced. Taken together, the identity of the C1/C72 basepair influenced the efficiency of cleavage irrespective of substrate context. The data further suggested that the guanosine at C1 plays an important role;

however, the base at the cleavage site (at C1) is not essential for cleavage to occur. Cleavage site recognition as function of changing GC1/CC72 In keeping with our previous reports pATSer variants carrying CK1 were miscleaved at the K1 position while replacement of CK1 with UK1

575

RNase P Cleavage and tRNA Precursors Processing

Table 1. Compilation of the different pATSer derivatives used in the present study Substrate pATSerUGG/C pATSerUGIno/C pATSerUGA/U pATSerUGDMA/U pATSerUG2AP/U pATSerUGDAP/U pATSerUGPu/U pATSerUGIsoG/C pATSerUGdG/C pATSerUGc7dG/C pATSerUGRib/C pATSerUGdD/C pATSerUGU/C pATSerUGU/Ac pATSerUGU/DAPc pATSerCGG/C pATSerCGIno/C pATSerCG2AP/C pATSerCG2AP/U pATSerCGDAP/U pATSerCGA/U pATSerCGdG/C pATSerCGc7dG/C pATSerCGdD/C pATSerCGU/Ac pATSerCGU/DAPc pATSerUAG/C

Identity of residue at the K1 position

Identity of the C1/C 72 base-pair

Substrate cleaved with Pb2C

UK1 UK1 UK1 UK1 UK1 UK1 UK1 UK1 UK1 UK1 UK1 UK1 UK1 UK1 UK1 CK1 CK1 CK1 CK1 CK1 CK1 CK1 CK1 CK1 CK1 CK1 UK1

GC1/CC72 InoC1/CC72 AC1/UC72 DMAC1/UC72 2APC1/UC72 DAPC1/UC72 PuC1/UC72 IsoGC1/CC72 dGC1/CC72 c7dGC1/CC72 RibC1/CC72 dDC1/CC72 UC1/CC72 UC1/AC72 UC1/DAPC72 GC1/CC72 InoC1/CC72 2APC1/CC72 2APC1/UC72 DAPC1/UC72 AC1/UC72 dGC1/CC72 c7dGC1/CC72 dDC1/CC72 UC1/AC72 UC1/DAPC72 GC1/CC72

Ca Ca Ca K Ca Cb Ca Cb Ca Ca Ca K Ca Ca Ca Ca Ca K K Ca Ca K K Ca Ca Ca K

Comment on modification Missing 2NH2 Missing 2NH2 Two -CH3 at position 6 Missing O6 2NH2 and 6NH2 Missing 2NH2 and O6 O2 and 6NH2 2 0 OH replaced with 2 0 H Modifications: 2 0 H and c7 Missing 2NH2 2 0 H and the base is missing

Missing 2NH2 Missing O6 Missing O6 2NH2 and 6NH2 2 0 OH replaced with 2 0 H Modifications: 2 0 H and c7 2 0 H and the base is missing

All substrates except pATSerUAG/C carry a G at the C73 position. The underlined residues correspond to the C1/C72 base-pair in the substrate. a Data reported in and taken from Kikovska et al.21 b E. Kikovska (unpublished data). C, Have been subjected to Pb2C-induced cleavage; K, have not been subjected to Pb2C-induced cleavage. c These substrates were studied by Kikovska et al. 2 and experimental data are included in this study for comparison. See also Figure 1. Abbreviations, see the legend to Figure 1.

suppressed the frequency of miscleavage (Figure 3).2,19,20 (The data presented here are based on experiments performed under single turnover conditions at pH 6.0 as outlined in Materials and Methods.) In the CK1 context, replacing GC1 with purines lacking the exocyclic amine at position 2 of guanosine (2NH2) resulted in a significant increase in miscleavage while deleting O6 (i.e. 2APC1/UC72) only gave a small effect. Substituting 2 0 OH of GC1 and N7 (with 2 0 H at C1) did not influence cleavage site recognition to any significant extent (data not shown). Only a modest increase in miscleavage was observed for the UK1 variants AC1/UC72 and IsoGC1/CC72 compared to that observed in cleavage of the GC1/CC72 variant (Figure 3, the values for the UC1 derivatives are taken from Kikovska et al.2 for comparison). For most of the other variants we were not able to detect cleavage at K1 under these conditions (data not shown). The reason for the observed difference comparing miscleavage of the CK1 versus the UK1 variants is at present not clear, but it might be related to the fact that C at K1 is base-paired to GC73 in the substrate and/or the nature of “A248/NK1 interaction, i.e. A248/CK1 versus A248/UK1.2,16,20 Moreover, deleting the base (in the context of having a 2 0 H at C1) while keeping the phosphate backbone intact (dDC1/CC72) resulted

in substantial miscleavage at several positions (at K2, K1 and C2) in addition to cleavage at C1 (Figure 3). This was observed irrespective of context, i.e. CK1 or UK1 (data not shown for the CK1 variant). We conclude that GC1 is not absolutely necessary for cleavage at C1 but its presence suppresses Table 2. Summary of kcat/KM values as determined under multiple turnover conditions for different pATSer substrates carrying changes at C1/C72 by wild-type M1 RNA as indicated Substrate

kcat/KM (minK1 mM)

pATSerCGG/C pATSerCG2AP/C pATSerCG2AP/U pATSerCGDAP/U pATSerCGIno/C pATSerCGA/U pATSerCGdG/C pATSerCGc7dGC pATSerCGdD/C pATSerCGU/A pATSerCGU/DAP pATSerUAG/C

3.1G0.02 0.07G0.002 0.16G0.07 0.19G0.027 0.14G0.04 0.074G0.036 0.22G0.06 0.14G0.014 0.00019G9.5!10K5 0.0021G0.0001a 0.19G0.03a 8.6G2.8

kcat and Km values determined under multiple turnover conditions (see Materials and Methods) were used to calculate the kcat/Km values and each value is an average of at least three independent experiments and is given as a mean valueGthe deviation of this value. a Data taken from Kikovska et al.2

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RNase P Cleavage and tRNA Precursors Processing

2NH2 of GC1 influences Mg2C binding A recent report where we studied RNase P RNAmediated cleavage as a function of replacing GC1/ CC72 with UC1/AC72 indicated the importance of the structural architecture of the C1/C72 base-pair for Mg2C binding in its vicinity.2 Based on this we decided to study the rate of cleavage under single turnover conditions at pH 6.0, where chemistry is suggested to be rate-limiting (see above), as a function of [Mg2C]. Importance of the 2NH2 of GC1 Figure 2. Histogram showing the rate of cleavage of various pSu3 derivatives carrying substitutions at C1/C 72 as indicated. The rates were determined at pH 7.2 in buffer A at 100 mM Mg2C and at 37 8C (see Materials and Methods) using 0.11 mM substrate and 0.24 mM M1 RNA. The reaction times were adjusted to ensure that the rates were determined in the linear part of the curve of kinetics. The rates of cleavage are expressed as percent of cleavage per min as indicated and are averages of several independent experiments and the experimental errors are given as error bars. G/C, G/U etc indicate the identities of the residues at positions C1 and C72 in pSu3 (see Figure 1), i.e. the C1/C72 pair.

cleavage at other positions (see also Discussion). Moreover, 2NH2 of GC1 plays an essential role in the cleavage site recognition process in cleavage of the model hairpin substrate in particular for substrates with C at K1. By contrast the 2 0 OH and N7 (at least not for a substrate with 2 0 H at C1) of GC1 does not appear to play any significant role in cleavage site recognition.

As shown in Figure 4(a), deleting 2NH2 of GC1 or disruption of the ring structure (the RibC1/CC72 variant) resulted in a distinct reduction in the rate of cleavage at lower Mg2C concentrations while at higher concentrations the difference in rate relative to that for the wild-type (GC1/CC72) was less pronounced. This indicated that addition of Mg2C could at least partly rescue the phenotype caused by the deletion of 2NH2 at the RNase P cleavage site. In keeping with this the Mg2C profiles for the 2APC1/ UC72 and DAPC1/UC72 variants were very similar compared to that for the wild-type, i.e. GC1/CC72 (Figure 4(b)). Both these variants carry 2NH2 (Figure 1). Optimal cleavage of IsoGC1/CC72 (where 2NH2 has been replaced with O; Figures 1 and 4(a), and data not shown) required higher Mg2C concentration, similar to what we observed for cleavage of substrates lacking 2NH2. Together, these data suggested that 2NH2 of GC1, present in the shallow (minor) groove (see also Discussion), influences Mg2C binding likely in the vicinity of the cleavage site in the RNase P RNA substrate (RS)-complex. Moreover, we noted that introduction of bulky (two CH3) groups in the deep (major) groove in the substrate at the cleavage site also affected the Mg2C requirement (DMAC1/UC72, Figure 4(a)). Importance of the 2 0 OH and N7 of GC1

Figure 3. Miscleavage of various pATSer substrates carrying substitutions at C1/C72 as indicated where for example CGG/C corresponds to substrate pATSerCGG/C that carries C K1 , G C73 and G C1 /C C72 . In case miscleavage was observed at several positions the frequencies of cleavage at these positions were added and used to calculate the frequency of miscleavage. The data shown are averages of at least three independent experiments. The experiments were performed under single turnover conditions at pH 6.0 as outlined in Materials and Methods.

For the UK1 variant dGC1/CC72 an approximately twofold difference in rate was observed compared to cleavage of wild-type (GC1/CC72) irrespective of Mg2C concentration (Figure 4(c)). Replacing N7 with c7 did not change the Mg2C profile relative to that of the dGC1/CC72 variant up to 160 mM Mg2C, except that c7dGC1/CC72 was cleaved with ztwofold lower rates compared to cleavage of dGC1/CC72 (at 200 mM Mg2C no difference comparing rates of these two substrates was detected; Figure 4(c)). Thus, it appears that neither the 2 0 OH nor N7 (in the context of 2 0 H at C1) of GC1 affected the Mg2C requirement in cleavage of the model hairpin substrate pATSer, at least not to any significant extent. From our data it is also apparent that replacing UK1 with CK1 changes the Mg2C requirement dramatically such that a significantly higher Mg2C

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RNase P Cleavage and tRNA Precursors Processing

Contribution of the 2NH2 of GC1 to ground state binding To understand the role of the 2NH2, the 2 0 OH and N7 of GC1 with respect to ground state binding we determined the apparent binding constant appKd for the different substrates at two different Ca2C concentrations, 40 mM and 160 mM. The data are summarized in Table 3. Contribution of the 2NH2 of GC1 At low concentration (40 mM) the appKd were not changed more than tenfold compared to the wild-type GC1/CC72 (with the exception of RibC1/ CC72 and DMAC1/UC72). The general trend was that substrates lacking the 2NH2 bound with less affinity. Consistent with our previous data,2 increasing the Ca2C concentration resulted in decreased appKd values except for the substrate where the base at C1 had been deleted in the dGC1/ CC72 variant (i.e. dDC1/CC72). In this case appKd was not affected (Table 3). Further, for the UK1 variants no difference comparing appKd values for the wild-type GC1/CC72, InoC1/CC72 and 2APC1/UC72 was observed at 160 mM Ca2C while for RibC1/CC72 and PuC1/CC72 compared to GC1/CC72 the difference was less pronounced. Moreover, the appKd value for IsoGC1/CC72 is significantly higher (30-fold at 160 mM Ca2C) compared to that for the wild-type

Table 3. Apparent Kd values for various pATSer derivatives and wild-type M1 RNA at 40 and 160 mM Ca2C as indicated Figure 4. M1 RNA-mediated cleavage of various pATSer derivatives as indicated as a function of Mg2C concentration under single turnover conditions at pH 6.0 in buffer C at 37 8C (see Materials and Methods). The concentrations of M1 RNA and substrate were 3.2 mM and !10 nM, respectively. The time of incubation was adjusted to ensure that the measurements were done in the linear part of the curve of kinetics. For the calculations we used the 5 0 cleavage fragment. The given data are the average of three independent experiments. The Mg2C profile for the wild-type substrate pATSerUGG/C is shown in (a), (b) and (c) for reference. (a) Cleavage of substrates with substitution at position 2 in the base (see Figure 1). (b) Cleavage of substrates with substitution at position 6 in the base. (c) Cleavage of substrates with substitutions of the 2 0 OH at C1, N7 and as a result of deleting the base at C1.

concentration is required to reach plateau for the CK1 variants (data not shown) in keeping with our previous findings.2 This might be influenced by that CK1 base-pairs with GC73 in the substrate and/or that the structural topography of the “A248/NK1 interaction” is changed in the case of the CK1 substrate derivatives (for further discussion see Kikovska et al.2).

Substrate

appKd (mM) 40 mM

appKd (mM) 160 mM

pATSerUGG/C pATSerUGIno/C pATSerUGA/U pATSerUGDMA/U pATSerUG2AP/U pATSerUGDAP/U pATSerUGPu/U pATSerUGIsoG/C pATSerUGdG/C pATSerUGc7dG/C pATSerUGRib/U pATSerUGdD/C pATSerUGU/C pATSerUGU/A pATSerUGU/DAP pATSerCGG/C pATSerCGIno/C pATSerCG2AP/U pATSerCGDAP/U pATSerCGA/U pATSerCGdG/C pATSerCGU/A pATSerCGU/DAP

0.11G0.056 0.81G0.27 0.35G0.19 4.6G0.9 0.15G0.053 0.03G0.008 1.3G0.33 nd 0.12G0.011 0.16G0.04 2.0G0.61 0.68G0.19 nd 1.3G0.078a 4.8G1.2a 11G7a nd nd nd nd nd nd nd

0.014G0.002 0.020G0.0033 0.041G0.014 0.12G0.01 0.012G0.004 0.027G0.004 0.037G0.014 0.43G0.08 0.04G0.02 0.003G0.0009 0.09G0.015 0.69G0.11 0.29G0.12 0.13G0.023a 0.73G0.49a 0.24G0.06a 1.8G0.48 0.43G0.12 0.28G0.11 2.0G0.58 0.065G0.02 3.2G0.38a 0.40G0.077a

The experiments were performed as described in Materials and Methods. The underlined residues correspond to the C1/C72 base-pair in the substrate. nd, not determined. Each value is an average of several independent experiments. a Values taken from Kikovska et al.2

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RNase P Cleavage and tRNA Precursors Processing

(GC1/CC72). For the CK1 variants we also observed an increase in appKd in those cases where the 2NH2 was missing (Table 3). We conclude that the 2NH2 of GC1 contributes to ground state binding irrespective of the nature of the A248/NK1 interaction.

Importance of the 2 0 OH and N7 of GC1 The appKd value for dGC1/CC72 (compare values for GC1/CC72 and dGC1/CC72) revealed that it appears that the 2 0 OH of GC1 does not contribute to ground state binding to any significant extent irrespective of Ca2C concentration. Nor did we observe any change in appKd as a result of replacing N7 of GC1 in the dGC1/CC72 variant with c7 (compare dGC1/CC72 and c7dGC1/CC72 variants) at 40 mM while a tenfold increase in binding affinity was detected at the higher concentration comparing these two deoxy substrate variants.

Finally, based on appKd values for AC1/UC72, DAPC1/UC72 and 2APC1/UC72 it appeared that the presence of exocyclic amines both at position 2 and 6 increased the affinity in particular at the lower divalent metal ion concentration. This is in contrast to when two methyl groups were added to 6NH2 of AC1 (i.e. DMAC1/UC72) where a significant decrease in appKd was observed (Table 3; compare values for GC1/CC72, AC1/UC72 and DMAC1/UC72). The 2NH2 of GC1 influences the rate of cleavage under saturating single turnover conditions Next we determined the rate of cleavage (kobs) for the different C1/C72 variants at pH 6.0 under saturating single turnover conditions. At this pH chemistry is suggested to be rate-limiting (see above). The kobs and kobs/Ksto (Zkcat/Km) were determined at both 40 mM and 160 mM Mg2C and the results are summarized in Table 4.

Table 4. Summary of cleavage rate constants in cleavage of pATSer derivatives under saturating single turnover conditions at pH 6.0 and different Mg2C concentrations as indicated [Mg2C] 40 mM kobs

Substrate pATSerUGG/C pATSerUGIno/C pATSerUG2AP/U pATSerUGDAP/U pATSerUGA/U pATSerUGDMA/U pATSerUGIsoG/C pATSerUGPu/U pATSerUGdG/C pATSerUGc7dG/C pATSerUGRib/C pATSerUGdD/C

a

pATSerUGU/A pATSerUGU/C

pATSerUGU/DAPa pATSerCGG/C pATSerCGIno/C pATSerCG2AP/C pATSerCGDAP/U pATSerCGA/U pATSerCGdG/C pATSerCGU/Aa pATSerCGU/DAPa

C1 C1 C1 C1 C1 C1 C1 C1 C1 C1 C1 K2 K1 C1 C2 C1 C1 C2 C1 K1 C1 K1 C1 K1 C1 K1 C1 C2 K1 C1 K1 C1 K1 C1 K1 C1

160 mM sto

kobs/K

3.0G0.43 0.17G0.013 1.9G0.12 3.0G0.20 0.36G0.06 0.04G0.05 nd 0.13G0.03 1.3G0.11 0.88G0.13 0.23G0.019

1.2G0.05 0.055G0.01 1.1G0.22 3.2G0.31 0.13G0.01 0.0042G0.0005 nd 0.05G0.01 1.8G0.26 0.41G0.083 0.04G0.003

nd

nd

0.0077G0.0039 nd nd 0.0005G0.00005

0.11G0.05 nd nd 0.015G0.004

nd

nd

nd

nd

nd

nd

nd

nd

nd

nd

nd

nd

nd

nd

nd

nd

kobs

kobs/Ksto

8.4G0.3 3.4G0.42 5.2G0.62 7.1G0.23 4.0G0.09 1.4G0.1 0.13G0.04 1.4G0.02 4.0G0.30 1.5G0.015 6.1G0.26 0.00049G33!10-5 0.00023G15!10-5 0.00059G26!10-5 0.00063G42!10-5 0.10G0.023 0.14G0.01 0.47G0.05 0.016G0.002 0.18G0.04 2.8G0.46 0.04G0.01 0.08G0.02 0.17G0.01 1.8G0.42 0.018G0.001 0.17G0.014 0.017G0.002 0.11G0.006 0.30G0.0023 0.18G0.04 1.4G0.46 0.013G0.001 0.016G0.002 0.015G0.001 0.041G0.002

13G1.6 4.1G0.43 9.7G1.2 15G0.44 5.8G0.17 0.49G0.07 0.032G0.005 4.4G0.32 9.1G1.4 3.9G0.61 3.9G0.14 0.000075G5!10K5 0.000078G5!10K5 0.00015G10!10K5 0.0002G13!10K5 nd 0.04G0.005 0.13G0.02 nd 0.077G0.016 1.2G0.03 0.02G0.002 0.04G0.007 0.06G0.003 0.63G0.022 0.013G0.0014 0.11G0.01 0.013G0.0015 0.018G0.0016 0.041G0.0041 0.077G0.016 0.89G0.12 nd nd nd nd

The experiments were performed as described in Materials and Methods. kobs/KstoZkkcat/Km; nd, not determined; K2, K1,C1 and C2 indicate the cleavage site. Each value is an average of several independent experiments. kobs and kobs/Ksto are expressed as minK1 and minK1!mMK1, respectively. Underlined residues correspond to the identity of the C1/C72 base-pair. a Values were taken from Kikovska et al.2

579

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Contribution of the 2NH2 of GC1 Comparing kobs for cleavage of GC1/CC72 with the values for InoC1/CC72, AC1/UC72, RibC1/CC72 and PuC1/CC72, which all lack the 2NH2, revealed an apparent reduction in kobs at the lower Mg2C concentration. For the UK1 variants the “introduction” of the missing exocyclic amine resulted in almost the same kobs as observed for the wild-type i.e. GC1/CC72 (Table 4; compare kobs for 2APC1/ UC72 and DAPC1/UC72 with those obtained in cleavage of GC1/CC72, InoC1/CC72, AC1/UC72, RibC1/CC72 and PuC1/CC72). We also observed that the difference in kobs detected at 40 mM Mg2C was reduced at the higher Mg2C concentration. Moreover, replacing G with IsoG at C1 resulted in an almost 100-fold reduction in kobs at 160 mM Mg2C. Together these data indicated the importance of the 2NH2 of GC1 with respect to catalysis conceivably by influencing Mg2C binding and/or by mediating an interaction between the substrate and M1 RNA (see Discussion). The contribution of 2NH2 at the cleavage site to catalysis was further supported by the kobs data demonstrating that the absence/presence of this chemical group resulted in a decreased kobs value relative to wild-type GC1/CC72 also in the case where UK1 had been replaced with CK1 (Table 4; compare kobs for cleavage of the pATSerCG variants GC1/CC72, InoC1/CC72, 2APC1/CC72 and AC1/ UC72). However, we noted that having NH2 both at position 2 and 6 showed a distinct and clear difference in kobs comparing the UK1 and CK1 variants with DAPC1/UC72 indicating the complexity in orchestration of chemical groups to ensure efficient cleavage. The underlying reason to this difference is at present not clear. We conclude that the 2NH2 of GC1 contributes to catalysis irrespective of the nature of the A248/NK1 interaction. It also appears that the presence of oxygen at position 2 on the base inhibits cleavage (compare values for IsoGC1/CC72, AC1/UC72 and GC1/CC72; Table 4). Influence of 6NH2 at C1 on catalysis Introduction of two methyl groups at position 6 of adenosine (DMAC1/UC72) also affected kobs in particular at 40 mM Mg2C (Table 4; compare DMAC1/UC72, AC1/UC72 and GC1/CC72). But the effect was not as large as the change observed in appKd, suggesting that the presence of the methyl

groups at position 6 mainly influenced ground state binding (see above; Table 2). Influence of the 2 0 OH and N7 of GC1 on catalysis The kobs values for the dGC1/CC72 and c7dGC1/ CC72 variants revealed a twofold effect as a result of substituting the 2 0 OH with 2 0 H and an additional twofold reduction due to replacement of both N7 and the 2 0 OH of GC1 (i.e. c7dGC1/CC72) irrespective of Mg2C concentration. This indicated that the 2 0 OH and N7 (in the 2 0 H context) of GC1 only play minor roles with respect to catalysis at least in the U K1 context. Finally, as indicated above the presence of the base at position C1 is not required for cleavage to occur. However, the rate of cleavage at C1 was reduced O four orders of magnitude, indicating the importance of the presence of the base at C1 for catalysis (Table 4; compare cleavage of dGC1/CC72 and dDC1/CC72). The absence of 2NH2 at position C1 affected the charge distribution at the cleavage site In a previous report we substituted the 2 0 OH with 2 NH2 (referred to as 2 0 N) at the cleavage site, i.e. at K1 (Figure 1). As a result, cleavage at K1 was observed in addition to cleavage at the canonical site C1. The frequency of cleavage at K1 increased with decreasing pH most likely due to that the 2 0 N becoming protonated at lower pH (the pKa value of the 2 0 N of dinucleotides has been determined to be 6.0–6.2).24,25 The positive charge at the canonical cleavage site was suggested to prevent cleavage at this position. Hence, we suggested that by studying the cleavage pattern as a function of pH the 2 0 N at the cleavage site could function as a probe to identify factors influencing the charge distribution at the cleavage site, i.e. the canonical cleavage site C1.20 We used this approach to investigate whether the structural architecture of the C1/C72 influenced the charge distribution at the cleavage site by studying the cleavage pattern of 2 0 N substituted substrates at different pH. Thus, we generated pATSerUK1GC73 derivatives with 2 0 N at position K1 and GC1/CC72, InoC1/CC72, 2APC1/UC72, DAPC1/CC72 or AC1/UC72. The different variants together with the corresponding all-ribo substrates were subjected to cleavage under single turnover conditions as a function of pH. As shown in Figure 5 the presence of 2NH2 (i.e. the exocyclic amine) at position C1 resulted in a shift of the cleavage site 0

Figure 5. Cleavage of different pATSerUG derivatives with 2 0 NH2 at the K1 position and different substitutions at C1/C72 by M1 RNA at different pHs as indicated. Reactions were done under single turnover conditions at 37 8C in buffer B at different pHs (5.5, 6.1, 7.0 and 8.4) in the presence of 160 mM Mg2C as described20 (see also Materials and Methods). The concentrations of M1 RNA and substrate (S) were 3.2 mM and !10 nM, respectively.

580 from K1 to C1 at a lower pH compared to cleavage of the substrates lacking the 2NH2. (We also noted that in particular the 2 0 N substituted InoC1/CC72 variant was cleaved at a low frequency at the C2 position (data not shown). This was most apparent at higher pH but cleavage was not further characterized.) For the all-ribo substrates we observed no change (except for pATSerUGA/U where a twofold increase was detected) in the frequency of cleavage at the K1 position with increasing pH (data not shown). We conclude that the charge distribution at the cleavage site is affected by the presence of the exocyclic amine in the shallow groove at the C1 position.

Discussion tRNA precursors are the major class of substrates for RNase P in the cell while the precursor to 4.5 S RNA represents another class of RNase P substrates, viz., RNA hairpin-loop substrates.1 Most tRNA precursors carry a G immediately downstream of the RNase P cleavage site.15 Previous work using various tRNA precursors indicated the importance of guanosine at the cleavage site with respect to cleavage site recognition and efficiency of cleavage under physiological pH conditions.4,5,7,8,26–28 Moreover, the influence of the presence of GC1/CC72 on substrate binding and rate of cleavage has been documented both for E. coli and Bacillus subtilis RNase P.2,10 Based on data where we studied precursors where GC1/CC72 had been replaced with UC1/AC72 we recently concluded that the C1/C72 base-pair in a tRNA precursor plays an important role in substrate discrimination by RNase RNA.2 In the present study we showed that replacement/deletion of chemical groups of GC1 at the RNase P cleavage site affected cleavage site recognition, ground state binding and rate of cleavage under conditions where chemistry is suggested to be rate-limiting. Our data further indicated the importance of the exocyclic amine (2NH2) of GC1 in these steps and thus extend our previous findings that indicated the importance of the 2NH2 at the cleavage site in cleavage site recognition in cleavage of tRNAHis and tRNA model precursor substrates.9,13 However, the presence of the base at C1 is not essential for cleavage but its presence is suggested to suppress cleavage at other positions or in other words its absence unmasks alternative cleavage sites due to the change in the local structural topography at C1 (this work). The Pb2C-induced cleavage patterns of pATSerCGG/C and pATSerCGdD/C were reported to be very similar (see also Table 1).21 Hence, we consider it unlikely that cleavage at the alternative sites is due to the deletion of the base at C1 causing an overall structural change of the substrate. Rather we favor the model suggesting that the G at C1 acts as a guiding nucleotide for RNase P.8,12 Taken together, our findings provide one rationale to why most tRNAs carry a G at their

RNase P Cleavage and tRNA Precursors Processing

5 0 end, viz., the contribution of GC1 (and the 2NH2) to RNase P processing of the corresponding precursor. Function of the 2NH2 of G at the RNase P cleavage site The data presented here suggest that the 2NH2 at the cleavage site (at C1) influences substrate binding, rate of cleavage and cleavage site recognition. Moreover, we observed that its absence changed the Mg2C profile and the charged distribution at the cleavage site. In addition, our recent data indicated that the 2NH2 at C1 in the substrate influences catalysis most likely by affecting the positioning of the metal(II) ion(s) responsible for generating the nucleophile.21 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 Mg2C in the vicinity of the cleavage site. The 2NH2 is exposed in the shallow (minor) groove making it accessible for hydrogen bonding in the formation of the RS-complex (at least initially). Cross-linking data suggest that GC1 of tRNA is positioned in the vicinity of in particular residues A248, A249 and A330–A333.29–31 This is in keeping with chemical modification and nucleotide analogue interference data that also indicate that these two regions are in close contact with the substrate.32–35 Thus, it is plausible that the 2NH2 of GC1 in the precursor substrate interacts via hydrogen bond formation with a residue(s) in the A248A249 and/or A330–A333 regions. In the recently published crystallographic structures of type A (Thermatoga maritima)36 and type B (Bacillus stearothermophilus)37 the A248A249 region is modeled to be located in the vicinity of residue C1 in the substrate. Available data suggest that residue A248 interacts with the residue immediately upstream of the cleavage site, i.e. at position K1 in the 5 0 leader of the precursor22 (see also other references18,38). Residue A249 pairs with the well-conserved G residue at position 30037 and substitution of either A249 or G300 influences the catalytic performance of RNase P RNA.39,40 Moreover, cleavage of C1/C 72 tRNATyrSu3 precursor variants using wild-type and G300 M1 RNA mutant derivatives appears to indicate that the catalytic performance for the G300 mutants is dependent on the nature of the C1/C72 base-pair (unpublished data). Thus, a possibility is that the A249G300 pair interacts with the GC1 residue in the substrate and that the 2NH2 plays an important role in the formation of this interaction. Based on the type A RNase P RNA structural data36 the authors argue that residues A66, A248 and A351 mark the active site making it possible, but not mutually exclusive, that the interaction between GC1 and RNase P RNA could also involve A66 or A351. In this context we note that from earlier studies it appears that efficient cleavage requires extensive denaturation of the amino acyl acceptor-stem of the precursor.41 Based on this we cannot exclude that the

581

RNase P Cleavage and tRNA Precursors Processing

Watson–Crick surface of GC1 is accessible for interaction with residue(s) in RNase P RNA at some stage during (but after initial binding) RS-complex formation. Cleavage of substrates lacking 2NH2 at C1 required higher Mg2C concentration (this work) suggests that the possible interaction between RNase P RNA and 2NH2 of GC1 is mediated by Mg2C (i.e. acting as an outer sphere ligand, see below) and/or influences Mg2C binding at the cleavage site as outlined in Figure 6. The importance of Mg2C is supported by previous data demonstrating that replacement of Mg2C with Ca2C influences cleavage site selection for a tRNA precursor with inosine at the cleavage site.9,42 Moreover, although exocyclic amines are unlikely used as ligands for Mg2C it has recently been reported that exocyclic amines, including 2NH2 of guanosine, are used as outer sphere contacts in the large ribosomal subunit from Haloarcula marismortui.43,44 Thus, at present we cannot exclude the possibility that the 2NH2 of GC1 acts as an outer sphere ligand for Mg2C at the cleavage site. Influence of the 2 0 OH of GC1 Substitution of the 2 0 OH at C1 with 2 0 H in a tRNA precursor as well as in an in vitro selected substrate in the B. subtilis system, a type B RNase P RNA, 45,46 only resulted in small effects on substrate binding and rate of cleavage under conditions where chemistry is suggested to be rate-limiting.10,47 Likewise, under similar conditions using derivatives of a small RNA hairpin model substrate and M1 RNA (a type A RNase P RNA) we did not detect any significant effect on ground state binding or rate of cleavage (Tables 3 and 4).2,20 Thus, under conditions where chemistry is suggested to be rate-limiting the 2 0 OH at C1 influences cleavage moderately irrespective of type of RNase P RNA (A or B) or substrate. This is in

keeping with our previous report where we studied cleavage of 2 0 deoxy substituted model substrates mimicking the tRNAHis precursor cleavage site.38 However, previous data suggested that the 2 0 OH at the C1 position affects cleavage efficiency for M1 RNA Otenfold under multiple turnover conditions where the rate of cleavage is not rate-limiting, i.e. pH R7.5.12,48 This is in agreement with our findings when we analyzed cleavage of some of the CK1 derivatives under multiple turnover conditions at pH 7.2: kcat/Km decreased Otenfold due to replacement of the 2 0 OH at C1 (Table 2). Under multiple turnover conditions at pH 7.2 the 2 0 OH therefore appears to play a role in the cleavage reaction. The reason for the different conclusions based on saturating single turnover data generated under conditions where chemistry is rate-limiting and data generated under multiple turnover conditions is not clear but might indicate that the 2 0 OH at C1 plays a role in, for example, release of the product. Nonetheless, these results emphasize the importance of studying cleavage under various conditions. Influence of N7 of GC1 The N7 group of G and A in RNA is exposed in the deep (major) groove. From a modification interference study using tRNA precursor derivatives it appears that introduction of a methyl group at the N7 position of GC1 interferes with RNase P cleavage. It was discussed that this either was due to an effect on substrate binding or the presence of a positive charge that influences Mg2C binding at the cleavage site.49 Here we observed only a modest effect on in particular the rate of cleavage as a result of replacing N7 in our model RNA hairpin substrate (pATSer) carrying a 2 0 H at C1. Thus, based on our data it appears that N7 plays a minor role in RNase P RNA-mediated cleavage after formation of the RS-complex, at least in the case of model RNA hairpin substrates with 2 0 H at C1. One possibility is

Figure 6. Model illustrating the canonical RNase P cleavage site. Only one Mg2C is shown; however, available data clearly indicate that more than one Mg2C is positioned in the vicinity of the cleavage site. Based on our present data the exocyclic amine (2NH 2, highlighted in gray) at position 2 of GC1 can act as an outer sphere ligand for one of these Mg ions or 2NH2 is involved in an interaction with RNase P RNA that influences the positioning of Mg2C in the vicinity of the cleavage site. In the Figure these alternatives are indicated by ?. a–c represent chemical groups at the cleavage site that have been suggested to act as ligands (outer or inner sphere) for Mg2C (for details see the text and as described by Kikovska et al.21 and references therein). The arrow marks the phosphorous atom that corresponds to the one to be attacked in RNase P RNAmediated cleavage. The interaction between residue 248 in M1 RNA and the residue at K1 corresponds to A248/NK1 interaction18,22 while the interaction between 294 in M1 RNA and the residue at C73 corresponds to the C73/294 interaction that is part of the RCCA-RNase P RNA interaction20,60 (and references therein).

582 that N7 of GC1 contributes, although to a small extent, to catalysis by influencing positioning of Mg2C at the cleavage site.20,50

Concluding Remark Guanosine plays an important role in RNAmediated cleavage of RNA also in other systems as exemplified by its role in the group I intron system where it pairs with UK1 establishing a G–U wobble pair at the 5 0 splice site. Moreover, the 3 0 OH of a guanosine acts as the nucleophile in the splicing reaction.14 In the group II intron cleavage reaction the invariant G of the AGC triad of domain 5 plays a critical role for catalysis: the 2NH2 contributes to binding while N7 and O6 are essential for the chemistry of the reaction.51 Likewise two guanosine bases are essential in cleavage mediated by the hairpin RNA that belongs to the small ribozyme. Guanosine at the cleavage site (GC1) plays a role in positioning the scissile bond and GC8 is suggested to be involved in the chemistry of cleavage.52–54 Here we showed that replacement/deletion of chemical groups of GC1 at the RNase P cleavage site influenced cleavage, in particular the exocyclic amine of GC1. But, in contrast to the group II system the 2NH2 at the RNase P cleavage site was shown to contribute both to ground state binding and rate of cleavage where the effect on the rate (kobs) was more accentuated compare to its effect on ground state binding. Together these data show the differential exploitation of guanosine in RNA-mediated cleavage of RNA.

Materials and Methods Preparation of substrates and M1 RNA The various pATSer derivatives were purchased from Dharmacon, USA or IBA GmbH, Germany. The RNA was purified, labeled at the 5 0 end and gel-purified according to standard procedures as described elsewhere.18,19,55 The M1 RNA variants were generated as run-off transcripts using T7 DNA-dependent RNA polymerase.55–57 Cleavage assay conditions M1 RNA activity was monitored under single turnover conditions in buffer A (50 mM Tris–HCl (pH 7.2), 5% (w/v) PEG 6000, 100 mM NH4Cl), in buffer B (50 mM Bis– Tris propane (pH 6.1 or as indicated), 5% PEG 6000, 100 mM NH4Cl) or in buffer C (50 mM Mes (pH 6.0 at 37 8C), 0.8 M NH4Cl) in the presence of MgCl2 or Mg(OAc)2 as indicated.2,20,21 The pH was adjusted with HCl (in the case of buffer C HOAc) and the given pH values were measured at 37 8C with all components added except M1 RNA and substrate. The concentrations of M1 RNA and substrates were R0.24 mM and %0.05 mM, respectively. All reactions were performed at 37 8C and the reaction products were separated on denaturing 20–22% (w/v) polyacrylamide gels and cleavage was quantified on a Phosphorimager (Molecular Dynamics 400S) as described19 (and references therein).

RNase P Cleavage and tRNA Precursors Processing

Determination of kcat/Km under multiple turnover conditions The kcat/Km values were obtained from determinations of the kinetic constants kcat and Km under multiple turnover conditions in buffer A at 160 mM Mg2C and pH 7.2 as described by Bra¨nnvall et al.18

Determination of the site of cleavage and the frequency of cleavage at different sites The site of cleavage was based on the mobility of the 5 0 cleavage fragments as described.16 The frequency of cleavage at different positions was quantified from the relative amounts of 5 0 cleavage products as a result of cleavage at the different positions as described elsewhere.16 These numbers were subsequently used to calculate the percentage of cleavage at indicated positions. Cleavage between residues K1 and C1 corresponded to the canonical RNase P cleavage site, i.e. at the C1 position while cleavage at other sites is referred to as miscleavage.

Binding assay conditions Spin columns were used to determine apparent equilibrium dissociation constants (appKd) in buffer C supplemented with 0.05% (w/v) Nonidet P40 and 0.1% (w/v) SDS in the presence of various concentrations of CaCl2 as described58,59 except that pre-incubation was 20 min and the time after mixing substrate with M1 RNA was 20 min. The substrate concentration was !10 nM and M1 RNA concentration was varied from 0.01 mM to 13.7 mM. appKd values were determined by non-linear regression analysis using Origin 7.0 software (Originlab) and the equation f c Zf t ![M1 RNA] free /(Kd C[M1 RNA]free), where fcZfraction of precursor substrate in complex with M1 RNA and ftZmaximum fraction of substrate able to bind M1 RNA, i.e. endpoint. Determination of the kinetic constants, kobs/Ksto and kobs, under single turnover conditions The kinetic constants kobs and kobs/Ksto (Zkcat/Km) were determined in buffer C at pH 6.0 in the presence of 40 mM and 160 mM Mg(OAc)2 as described.2,20 The final concentration of substrate was %40 nM while for M1 RNA the concentration was varied between 0.040 mM and 5.16 mM. For the calculations the 5 0 cleavage fragments were used and the time of cleavage for each substrate was adjusted to ensure that we were in the linear part of the curve of kinetics. The kobs and kobs/Ksto values were obtained by linear regression from Eadie–Hofstee plots.

Acknowledgements We thank our colleagues for discussions throughout this work. This work was supported by grants from the Swedish Research Council and the Swedish Strategic Research Foundation to L.A.K.

RNase P Cleavage and tRNA Precursors Processing

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Edited by J. Doudna (Received 11 January 2006; received in revised form 14 March 2006; accepted 18 March 2006) Available online 3 April 2006