Abstract
tRNA genes are transcribed as precursors and RNase P generates the matured 5' end of tRNAs. It has been suggested that residue − 1 (the residue immediately 5ʹ of the scissile bond) in the pre-tRNA interacts with the well-conserved bacterial RNase P RNA (RPR) residue A248 (Escherichia coli numbering). The way A248 interacts with residue − 1 is not clear. To gain insight into the role of A248, we analyzed cleavage as a function of A248 substitutions and N−1 nucleobase identity by using pre-tRNA and three model substrates. Our findings are consistent with a model where the structural topology of the active site varies and depends on the identity of the nucleobases at, and in proximity to, the cleavage site and their potential to interact. This leads to positioning of Mg2+ that activates the water that acts as the nucleophile resulting in efficient and correct cleavage. We propose that in addition to be involved in anchoring the substrate the role of A248 is to exclude bulk water from access to the amino acid acceptor stem, thereby preventing non-specific hydrolysis of the pre-tRNA. Finally, base stacking is discussed as a way to protect functionally important base-pairing interactions from non-specific hydrolysis, thereby ensuring high fidelity during RNA processing and the decoding of mRNA.
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Introduction
The tRNA genes are transcribed as precursors (pre-tRNA) and several enzymes are involved in the processing of pre-tRNA. Among these endoribonuclease P (RNase P) is responsible for generating tRNAs with matured 5ʹ ends. RNase P from all three kingdoms of life consists of both RNA and protein; in Bacteria, RNase P is composed of one RNA (RPR) and one protein subunit, C5. Irrespective of origin, the catalytic activity was thought to reside in the RPR1,2. Recent data, however, show that there exist RNase P activities solely based on proteins, (referred to as PRORPs) in human mitochondria, Arabidopsis thaliana, Trypanosoma brucei, in the algae Ostreococcus tauri and in Aquifex aeolicus3,4,5,6,7.
At high ionic strength, RPRs of different origins cleave pre-tRNA and a number of non-tRNA substrates efficiently at the correct site without proteins1,8,9,10,11,12. The RPR interacts with several regions of pre-tRNAs and model substrates. These are: the 3ʹ terminal RCC-motif (the RCCA-RNase P interaction, interacting residues underlined)13; and the T-stem/loop- (TSL) region of pre-tRNAs binds to the RPR TSL-binding site or TBS in the specificity (S) domain. In addition, the residue immediately 5ʹ of the cleavage site (N−1) is in close proximity to A248 (referred to as the A248/N−1 interaction and Escherichia coli numbering; Fig. 1). The crystal structure of bacterial (Thermotoga maritima) RNase P in complex with tRNA and recent cryo-EM structures of yeast, archaeal and human RNase P in complex with pre-tRNA (yeast) and tRNA suggest that in particular the TSL–TBS interaction is evolutionary conserved11,14,15,16,17,18,19,20,21, (see also22).
Illustration of the Eco RPR secondary structures. (A) Eco RPR secondary structure according to Massire et al.90. The heavy dashed demarcation line separates the S- and C-domains. The large gray box highlights the A248-region, and show the substitutions that were introduced at 248 (red arrows). The gray box in L15 marks residues that pair with the substrate 3ʹ end—the RCCA-RNase P RNA interaction (interacting residues underlined)12—in the RPR-substrate complex. The blue arrows and Roman numerals mark the Pb2+-induced cleavage sites as shown in Fig. 2 (black circles). The vertical line marked in blue marks the "332-region", which is also cleaved in the in presence of Pb2+(see also85,91). Residues highlighted with gray circles correspond to RNase T1 cleavage sites (see also Fig. 2, bands marked with red dots)92. The green dashed line and arrows mark the area in P18, which becomes accessible to RNase T1 cleavage upon on substitution of A248 with U (see Fig. 2, Eco RPRU248). (B) Sequence of alignment of the region which includes the conserved E. coli (Ec) A248, T. maritima (Tm93) A213, M. tuberculosis (Mtb28) A248 and the Archaea P. furiosus (Pfu9,12) A218, and neighboring sequences as indicated.
Residue A248 is well-conserved among bacterial RPRs. However, the nature of the A248/N−1 interaction is less clear. In the bacterial RNase P-tRNA co-crystal structure A248 is positioned close to the tRNA 5ʹ end18. This is also observed in the cryo-EM structure of an archaeal RNase P in complex with tRNA20. On the basis of biochemical and genetic data using E. coli (Eco) RPR, A248 has been proposed to form a cis Watson–Crick/Watson–Crick (cis WC/WC)23 pair with the N−1 residue in the substrate24,25. The identity of residue −1 in pre-tRNAs varies in E. coli and other bacteria, but in many of them it is a uridine26,27,28. We have provided data that this pairing does not correspond to a standard Watson–Crick pairing. Rather, the N−1 residue binds to a pocket where A248(wt) plays a central role, but it does not directly pair with the −1 residue16,29,30,31. Nucleotide analogue-modification interference studies further suggest that the Hoogsteen surface of A248 is important for a productive interaction with the substrate32. However, kinetic data argue against that N7 and 6NH2 of A248 form hydrogen bonds with the 2ʹOH and O2 (when present on the nucleobase, i.e. C or U) of residue −1, respectively, which both are oriented in the same direction in a structural model of the cleavage site31. Thus, the function of A248—and whether it interacts with residue –1—remains unclear. The crystal and cryo-EM structures of the RNase P-tRNA complexes (bacteria and archaea) do not provide guidance because these structures represent the post cleavage stage of the RNase P catalyzed reaction18,20. We therefore decided to revisit and investigate the interrelationship between residue −1 and A248. To achieve this, we studied cleavage of all ribo pre-tRNA and model hairpin loop substrates, carrying different nucleobases at position −1 with Eco RPR 248-variants.
Here we provide data that the identities of both residue N−1 in the substrate and residue 248 in the RPR influence cleavage site selection and rate of cleavage. However, our data do not support the model where the well-conserved residue A248(wt) forms a cis WC/WC pair with N−1. This was particularly apparent studying different substrates carrying 3-methyl U at the N−1 position. Our combined data support a model where the structural topology of the active site varies and depends on the identity of the nucleobases at, and in proximity to, the cleavage site and their potential to interact. As a consequence, this affects the positioning of Mg2+ that activates the water that acts as the nucleophile resulting in efficient and correct cleavage. In this scenario we suggest that, besides participating in the anchoring of the substrate, the role of A248 in wild type bacterial RPR, which stacks on the tRNA G+1/C+72 base pair, is to exclude bulk water from accessing the amino acid acceptor stem and thereby prevent non-specific hydrolysis/cleavage of the pre-tRNA.
Results
Substituting residue 248 has minor effects on the overall structure of Eco RPR
To investigate the role and contribution of the well-conserved A248 to Eco RPR mediated cleavage we used wild type Eco RPRA248(wt) and three 248 variants: Eco RPRC248, Eco RPRG248 and Eco RPRU248 (Fig. 1). The generation and catalytic performance of Eco RPRG248 has been reported elsewhere30,31, while the other two RPRs were generated as outlined in “Materials and methods”. As predicted on the basis of previous studies, the C248 and U248 variants were catalytically active24 (see below).
First, we inquired whether substitution of A248(wt) with any of the other nucleobases affected the structure of Eco RPR. On the basis of structural probing with Pb2+ and RNase T1 (which cleaves 3ʹ of single stranded G residues) we reported that the overall structures of Eco RPRA248(wt) and Eco RPRG248 are very similar31. This was also the case for the C248 variant [Fig. 2; cf. lanes 2 and 3 (Eco RPRA248(wt)) and lanes 11 and 12 (Eco RPRC248)]. By contrast, a U at 248 affected the structure such that G-residues between P15 and the P18-loop became accessible to RNase T1 [Fig. 1; cf. lanes 3 (Eco RPRA248(wt)) and 6 (Eco RPRU248)]. This suggested that a U at 248 influences the structural integrity of P18, which plays a role in connecting the C- and S-domain via the P8/P18-interaction (Fig. 1). Moreover, compared to Eco RPRA248(wt), exposure to RNase T1 resulted in the appearance of an additional weak cleavage product located between residues 276 and 292, in particular in the case of G248 (Fig. 2; bands marked with *). This might indicate a change in the structure in this region in response to mutating A248(wt), see also Ref.31. With respect to the Pb2+-induced cleavage patterns, we did not detect any apparent difference comparing the 248 variants [Fig. 2; cf. lanes 2 (A248(wt)), 5 (U248), 8 (C248) and 11 (G248)]. We conclude that substitution of A248 in wild type Eco RPR resulted in a small (if any) overall structural effect with the exception of U248 where a notable structural change was detected in P18.
Structural probing of Eco RPR. Probing the structures of the Eco RPR 248-variants with Pb2+ and RNase T1. Roman numerals and black circles refer to Pb2+-induced cleavage sites in Eco RPR (Fig. 1)31,85,91. Numbers and red circles correspond to the RNase T1 cleavage sites according to Guerrier-Takada and Altman92, see Fig. 1A. The vertical black lines mark the P18- and 332-region. The vertical black line "P18" marks the extra RNase T1 cleavage sites between 292 and 314 in the U248 variant. The reactions were conducted using 0.5 mM Pb(OAc)2 and RNase T1 as described in “Materials and methods”.
Catalytic performance as a function of replacing the well-conserved A248 in Eco RPR
Choice of substrates and experimental outline
The role of residues A248 in Eco RPRA248(wt) and −1 in the substrate has previously been analyzed using variants of a Bacillus subtilis tRNAAsp precursor24,25. From these studies, the authors proposed a model where the −1 residue in the substrate forms a cis Watson–Crick (WC) base pair with A248. The model predicts that (i) breakage of this interaction shifts cleavage from the correct to an alternative site (see below), and (ii) introduction of a compensatory change that restores the N−1/N248 pairing should increase (rescue) cleavage at the correct site. To test this model and to investigate the role of A248, we used N−1 derivatives of the E. coli tRNASerSu1 precursor, pSu113,33, and two well-characterized model hairpin loop substrates, pATSer and pMini3bp, both derived from pSu1 (Fig. 3)15,31,34,35,36. The pATSer substrates have the amino acceptor-stem and T-stem intact while pMini3bp lacks the T-stem, T-loop and part of the acceptor-stem. Two pATSer variants were used, the first has the original T-loop (e.g. pATSerUG where U and G correspond to the residues at −1 and " +73", respectively; numbering refers to the position in tRNA; Fig. 3). In the other, the T-loop is substituted with a GAAA-tetra loop (e.g. pATSerUGGAAA). The latter interacts differently with Eco RPRA248(wt); it increases cleavage at the alternative site between −2 and −1 (Fig. 3C; see below)15,30,37. The short model substrates pMini3bp all have three-base-pair short stems, capped with GAAA-tetra loops (e.g. pMini3bpUG). Importantly, pSu1 and pATSer can interact with the TBS-region (see above) upon Eco RPR substrate complex formation, while pATSer variants with GAAA-tetra loops and pMini3bp cannot (or interact differently) due to their sizes and/or the presence of the GAAA-tetra loop15,30,31,38. We introduced the natural ribonucleobases (A, C, G and U) at position −1 (N−1) in all four substrate variants. For pATSer and pMini3bp, we also used variants carrying chemically modified ribonucleobases at −1 and +73. Varying both residue −1 and +73 allowed us to investigate the importance of having nucleobases at −1 that can pair with residue +73 with different numbers of hydrogen bonds. To further investigate whether U−1 in the model substrates pairs with A248(wt) in Eco RPR we introduced a methyl group (3mU) at − 1 (Fig. 3E), which interferes with cis WC/WC pairing with A248(wt). Finally, we replaced the 2ʹOH with 2ʹNH2 (or 2ʹH) and varied the +1/+72 base pair in pATSerUG to probe the de-protonation of the 2ʹNH2 (charge distribution; see below) at the canonical cleavage site in the RPR-substrate complex as a function of N248 identity (Fig. 3)38,39,40.
Secondary structure of substrates used in the present study. (A) pSu1, (B) pATSerNN, (C) pATSerNNGAAA, (D) pMini3bp, (E) structures of nucleobases, and (F) cleavage of pATSerUG by the different Eco RPR 248 variants. Residues highlighted in gray were introduced to generate the different variants carrying alternative nucleobases at positions −1 and +73. The black boxes illustrate the changes that generated substrates carrying 2ʹNH2 and 2ʹH as well as substitutions of residues at positions +1 and +72. The canonical (correct) cleavage sites between residues N−1 and N+1 in the different substrates are marked with black arrows. The gray arrows mark the alternative cleavage sites between N−2 and N−1 (referred to as position −1, see text). The seven-base loop (B, marked in gray) in pATSerNN was replaced with a GAAA-tetra loop (C, marked in gray) to generate pATSerNNGAAA, see14,15. Panel (F): lane (L) 1, pATSerUG no RPR added; lane 2, cleavage of pATSerCGGAAA with Eco RPRA248(wt); lane 3, cleavage of pATSerUG with Eco RPRA248(wt); lane 4, cleavage of pATSerUG with Eco RPRC248; lane 5, cleavage of pATSerUG with Eco RPRG248; lane 6, cleavage of pATSerUG with Eco RPRU248. Sub, substrate and 5ʹCL Frags marks the migration of the 5ʹ cleavage products as a result of cleavage at +1 and −1. The reaction was performed in buffer C at 800 mM Mg2+ with 0.8 μM Eco RPR (irrespective of variant) and ≤ 0.02 μM substrate for 10 s as described in “Materials and methods”.
The Mg2+ concentration for optimal cleavage rates of pMini3bp substrates using Eco RPRA248(wt) and Eco RPRG248 is 800 mM; this is higher than for the other substrates15,30,31. Moreover, on the basis of our published data where we studied cleavage of pATSer and pMini3bp variants using Eco RPRA248(wt) and Eco RPRG248, we assumed that optimal cleavage rates are reached at 800 mM Mg2+ also for the other 248 variants15,30,31,37. To be able to directly compare the cleavage rates, we decided to perform all the experiments discussed below at 800 mM Mg2+. Also, at this Mg2+ concentration the likelihood of detecting cleavage increases, see e.g.,31. We emphasize that the C5 protein interacts with residues N−4–N−8 in the 5ʹ leader but not N−141,42 and that we were primarily interested in the catalytic performance of the RPR in the absence of C5. Hence, these studies were performed without the C5 protein.
Cleavage of the different substrates was studied with respect to (i) cleavage site recognition and (ii) rate of cleavage (single turnover; see “Materials and methods”). The canonical (also referred to as correct cleavage or the +1 position) site corresponds to cleavage between residues −1 and +1 (Fig. 3), while cleavage at other positions are referred to as alternative sites or miscleavage; e.g., cleavage at −1 relates to cleavage between −2 and −1 in the 5ʹ leader. The frequencies of cleavage at +1 are presented in Figs. 4, 6 and 8 while the rate constants (kapp), determined under single turnover conditions for the combinations discussed above, are shown in Tables 1, 2 and 3. For clarity and guidance, the experiments using different substrate/RPR (N248) combinations are referred to as "Experiment Series (ExpS)" in the figures and tables where 1.1 corresponds to substrate 1, pSu1, having A at −1 while 1.2 has C, i.e. pSu1A−1 and pSu1C−1, respectively, and substrate 2.1 pATSer having A−1 and G+73 (pATSerAG and A−1/G+73 substrate/variant) etc. In the first set of experiments we studied cleavage of the full-size pre-tRNASerSu1 (pSu1; ExpS 1.1–1.4) and pATSer N−1/N+73 (ExpS 2.1–2.8; Fig. 4A,B, and Table 1) variants that can interact productively with the TBS in the RPR S-domain (see above). These results are discussed below in “Substrates that can interact productively with the TBS-region—influence of changes of the nucleobase at −1 in substrates and residue 248 in the RPR”. Following this we analyzed the impact of N−1/N+73 variants in substrates that cannot form a productive interaction with the TBS, pATSerGAAA (ExpS 3.1–3.6; Fig. 4C and Table 1) and pMini3bp (ExpS 4.1–4.12; Fig. 4D and Table 2) variants. These data are discussed in “Substrates that cannot interact productively with the TBS-region—influence of changes of the nucleobase at −1 in substrates and residue 248 in the RPR”. In “Altering the WC-surface of a U at position −1 and influence of the N248 identity”, we discuss substrates carrying CH3 at position 3 on the nucleobase that alter the WC-surface of U−1 in model substrates (ExpS 5.1–5.3; Fig. 6 and Table 3). In each sections (A-C), we summarize the data with respect to the possible interaction between residues A248 and −1.
Frequencies of cleavage at +1 by Eco RPR 248 variants. Histograms summarizing frequencies of cleavage at +1 in % for the various substrate and Eco RPR 248 combinations as indicated. (A) Cleavage of pSu1(N−1) variants, Exp Series (ExpS) 1.1–1.4. (B) Cleavage of pATSer(N−1N+73) variants, Exp Series (ExpS) 2.1–2.8. (C) Cleavage of pATSer(N−1N+73)GAAA variants, Exp Series (ExpS) 3.1–3.6. (D) Cleavage of pMini3bpN−1/N+73 variants, Exp Series (ExpS) 4.1–4.12. To calculate the frequencies of cleavage at +1 we used the 5ʹ cleavage fragments and mean and experimental errors were calculated from at least three independent experiments.
Following this, in “Kinetic constants kobs and kobs/Ksto and activation energy as a function of N248 identity” we present single turnover kinetic data for cleavage of the model substrate pATSerUG with the different 248 variants. These experiments were performed at different temperatures with the objective to determine the activation energy as a function of the N248 identity. In the final “Differential effects due to replacement of the 2ʹOH at −1 with 2ʹH or 2ʹNH2 in pATSerUG and influence of the N248 identity on the charge distribution at the cleavage site”, we probe the influence of the N248 identity on the charge distribution at the cleavage site.
Substrates that can interact productively with the TBS-region—influence of changes of the nucleobase at −1 in substrates and residue 248 in the RPR
pSu1 variants (Fig. 4A and Table 1; ExpS 1.1–1.4): All the −1 variants were cleaved mainly at the +1 site and at the alternative position −1 (Fig. 4A), irrespective of the identity of residue 248. Except for U248, the U−1 variant (ExpS 1.4) was cleaved with the highest rate at +1 where kapp(+1) was highest for A248(wt). With respect to the wild-type substrate, pSu1C−1 (ExpS 1.2), A248(wt) was the most efficient catalyst; the kapp values for the other three variants were lower.
For pSu1A−1 (ExpS 1.1), changing Eco RPRA248(wt) to any of the other nucleobases resulted in decreased cleavage frequency at +1 relative to −1, while comparing kapp values for cleavage at +1 and −1 with the different RPRs differed ≤ two-fold. Cleavage of pSu1C−1 (ExpS 1.2) at +1 was reduced and increased at −1 using G248, but there was no apparent difference for the other 248 variants. The increased cleavage at −1 for G248 is also noticeable by comparing kapp(+1) and kapp(−1) values (Table 1). The wild-type A248 and G248 variant cleaved pSu1G−1 at +1 with slightly lower frequencies than C248 and U248 (ExpS 1.3). Moreover, kapp(+1) values for all 248 variants were similar (ExpS 1.3) while kapp for cleavage at −1 were higher for A248(wt) and G248 compared to C248 and U248. Finally, with pSu1U−1 (ExpS 1.4) we detected modest differences in cleavage frequency at +1 and kapp(+1) (at most 2.6-fold change comparing A248(wt) and U248) while no apparent change in kapp(−1) was detected irrespective of RPR. We also noted that the 248 variants cleaved the different pSu1 N−1 substrates with low frequencies at other positions in the 5ʹ leader upstream of site −1 (not shown). Noteworthy, in E. coli the wild-type pSu1 has a C at position −1, which pairs with the discriminator base G+73 (Fig. 3A) and the C−1/G+73 pairing influences cleavage efficiency and site selection, see e.g.27.
pATSer variants (Fig. 4B and Table 1; ExpS 2.1–2.8): Cleavage of the N−1 variants carrying A, C, G and U more or less mirrored the results with pSu1 (cf. ExpS 1.1–1.4 vs. 2.1–2.4). Overall G248 cleaved with the highest rates both at +1 (pATSerAG; ExpS 2.1) and at −1 (pATSerGG; ExpS 2.3). Moreover, having a purine at 248 gives a more efficient catalyst compared to when a pyrimidine is present at this position with the exception of the "pATSerCG/G248" combination (ExpS 2.2).
Specifically, first pATSerAG (ExpS 2.1) was cleaved with roughly the same frequencies at +1 by all 248 variants with the possible exception for C248, which cleaved this substrate both at +1 and −1 with a lower rate compared to the other RPR variants. Second, compared to the other RPRs G248 cleaved pATSerCG more frequently at −1. This is also reflected in the kapp values for cleavage at +1 and −1, while the G248 and C248 RPRs cleaved pATSerCG at +1 with the same rates (ExpS 2.2). These results are contradictory to the formation of cis WC/WC pairing between C−1 and G248 in the RPR-substrate complex. Third, in contrast there appeared to be suppression/rescue of cleavage of pATSerGG at −1 using C248 (comparing frequencies of cleavage at −1 and kapp(−1); Fig. 4B and Table 1; cf. ExpS 2.3 C248 vs. G248). Fourth, pATSerUG (cf. ExpS 2.4) was almost exclusively cleaved at +1 by all 248 variants (see also Fig. 3F). However, U248 cleaved pATSerUG with a significantly lower rate at +1 than the other RPR variants (Table 1).
For the pATSer variants with "unnatural" nucleobases at −1 and +73, reducing the number of potential hydrogen bonds between −1 and +73 from three to two restored cleavage at +1 for G248 to the level observed for the other 248 variants (Fig. 4B; cf. ExpS 2.2 and 2.5, i.e. pATSerCG vs. pATSerCIno). This appeared to be the result of an increase in the rate of cleavage at +1 while hardly any effect on the rate was detected for cleavage at −1 (Table 1; cf. ExpS 2.2 and 2.5). By contrast, the potential formation of three hydrogen bonds between U−1 and DAP+73 resulted in increased miscleavage for all four 248 variants (Fig. 4B; cf. ExpS 2.4 and 2.8, i.e. pATSerUG vs. pATSerUDAP). This was accompanied with noticeable rates of cleavage at −1 (Table 1; cf. ExpS 2.4 and 2.8). In keeping with this, the kapp values for cleavage at +1 were lower for pATSerUDAP relative to pATSerUG for all RPR variants.
In summary (see Fig. 5A), (i) when the T-loop can form a productive interaction with TBS in the S-domain we did not detect any conclusive evidence for cis WC/WC pairing between N−1 and N248. However, for some of the combinations, cis WC/WC pairing cannot be excluded (see also the “Discussion”). (ii) The potential to form three H-bonds between N−1 and N+73 affected both cleavage site selection and dependent on substrate-RPR combination the rate of cleavage. (iii) For Eco RPRA248(wt), cleavage of substrates with natural nucleobases at N−1, the U−1 substrates are preferred.
Summary of data for N−1/N248 cis WC/WC base paring. Boxes marked in gray are consistent with cis WC/WC base-pairing; light gray marks those combinations where one combination (or weak agreement/non-WC/WC pairing e.g. GU-pairing) are consistent with cis WC/WC base-pairing, e.g. cf. pSu1U−1/A248- vs pSu1A−1/U248-combinations. Boxes marked in red highlight the combinations that are not in agreement with cis WC/WC base pairing, while no color indicates other combinations. The grey ExpS boxes refer to the Experimental Series, e.g. 1.1–1.4 and 2.1–2.8 etc., as shown in Figs. 4 and 6, and Tables 1, 2 and 3. (A) Experiment series using pSu1 (ExpS 1.1–1.4) and pATSer (ExpS 2.1–2.8) variants, which can establish a productive interaction with the TBS region in the S-domain (see main text for details). (B) Experiment series using pATSerGAAA (ExpS 3.1–3.6) and pMini3bp (ExpS 4.1–4.12) variants, which cannot form a productive interaction with the TBS region in the S-domain (see main text for details). (C) Experiment series for model substrates with a 3-methyl group at U−1 (ExpS 5.1–5.3).
Frequencies of cleavage-site selection for 3-methylated substrates by Eco RPR 248 variants. Histograms summarizing frequencies of cleavage at +1 in % during Eco RPR-mediated cleavage of pATSer3mUG (ExpS 5.1), pATSer3mUGGAAA (ExpS 5.2) and pMini3bp3mUG (ExpS 5.3) as indicated. We used the 5ʹ cleavage fragments to calculate the frequencies of cleavage at +1; mean and experimental errors were calculated from at least three independent experiments.
Substrates that cannot interact productively with the TBS-region—influence of changes of the nucleobase at −1 in substrates and residue 248 in the RPR
pATSer GAAA-tetra loop variants (Fig. 4C and Table 1; ExpS 3.1–3.6): Replacement of the T-loop with a GAAA-tetra loop in the pATSer variants resulted in reduced frequency of cleavage at +1 for several combinations and lower kapp(+1) (cf. ExpS 2.1–2.4 vs. 3.1–3.4). However, dependent on substrate-RPR combination the decrease in rate at +1 varied between ≈ two- to 1000-fold; for example the kapp(+1) ratio for pATSerCG/pATSerCGGAAA and C248 showed a 1000-fold difference, but only ≈two-fold for pATSerCG/pATSerCGGAAA and G248 (Table 1; ExpS 2.2 vs. 3.2, cf. kapp 10 vs. 0.01 and 11 vs. 5, respectively). For cleavage at −1, the decrease in kapp(−1) was lower (≤ 20-fold) in response to substituting the T-loop with GAAA for all four 248 variants except for cleavage of pATSerGG vs. pATSerGGGAAA with C248. Here the decrease in (kapp(−1)) was > 100-fold (Table 1; ExpS 2.3 vs. 3.3). Moreover, for the "pATSerAGGAAA/C248", "pATSerCGGAAA/G248" and "pATSerGGGAAA/C248" combinations, we did observe significant cleavage at +1 with frequencies (relative to −1) comparable to those with an intact T-loop (e.g. cf. C248 cleavage of pATSerAG and pATSerAGGAAA; Fig. 4B,C; ExpS 2.1 vs. 4.1). Substitution of the T-loop with the GAAA-tetra loop in pATSerUG increased cleavage at −1 for U248, while we detected only small changes for the other 248 variants including the wild type (cf. Fig. 4B,C; ExpS 2.4 vs. 3.4). However, all RPR variants cleaved pATSerUGGAAA with measurable kapp(−1) values in contrast to pATSerUG (Table 1; ExpS 2.4 vs. 3.4). In keeping with the importance of pairing between N−1 and N+73 (see above) changing G to A at +73 in pATSerCGGAAA restored cleavage at +1 with increased rates irrespective of N248 variants (Fig. 4C and Table 1; cf. pATSerCGGAAA vs. pATSerCAGAAA, ExpS 3.2 and 3.5). By contrast, comparing kapp(+1) values for cleavage of pATSerUGGAAA and pATSerUAGAAA showed the opposite, i.e., lower kapp(+1) irrespective of 248 variant (Table 1; ExpS 3.4 vs. 3.6). For pATSerUAGAAA, increased frequency of cleavage at −1 was detected with U248 and it also cleaved pATSerUAGAAA at position −2 (≈20%) in the 5ʹ leader. Finally, a comparison of kapp(+1) values (and to some extent also kapp(−1)) suggested that A248(wt) and G248 were more efficient catalysts than C248 and U248 when pATSerNNGAAA variants were used.
pMini3bp variants (Fig. 4D and Table 2; ExpS 4.1–4.12): For the variants with natural nucleobases at −1 we did observe significant reduction in cleavage of the G−1 variant at +1 using A248(wt), G248 and U248 while C248 cleaved the G−1 substrate preferentially at +1 (Fig. 4D; ExpS 4.3). Moreover, irrespective of 248 variants pMini3bpCG and pMini3bpCA were cleaved mainly at +1 with some cleavage at −1 and cleavage of pMini3bpUA was detected only at +1 (Fig. 4D; ExpS 4.2, 4.3 and 4.5). The other three variants, pMini3bpAG, pMini3bpUG and pMini3bpUA (ExpS 4.1, 4.4 and 4.6), were cleaved almost exclusively at +1 by all four 248 variants.
With respect to the rate of cleavage we compared kapp(+1) (except for two substrates, see below) because the rates were significantly lower than for the other substrates, in particular at −1 (Table 2). Overall, kapp(+1) for A248(wt) and G248 were higher compared to C248 and U248; the highest kapp(+1) was for pMini3bpUG (ExpS 4.4) with A248(wt). This is in keeping with the trend seen with the other substrate variants, i.e. U−1 variants were in general cleaved with the highest rates at +1 (but cf. e.g., the "pATSerAG/G248" combination above). For the "pMini3bpCG/G248" combination (ExpS 4.2), kapp(+1) and kapp(−1) were both higher than when the other 248 variants, including A248(wt), were used. Comparing cleavage of pMini3bpCG vs. pMini3bpGG (ExpS 4.2 and 4.3) with G248, kapp(+1) was ≈1300-fold higher for pMini3bpCG, while kapp(−1) was ≈40-fold higher. No difference in kapp(+1) was detected for C248 cleaving these two substrates, while kapp(−1) was 3000-fold lower in cleaving pMini3bpCG than when G248 was used (Table 2; ExpS 4.2 and 4.3, cf. 0.0001 vs. 0.3). In fact, C248 was found to be a very poor catalyst with all pMini3bp substrates. Analyzing the (pMini3bpUG and pMini3bpAG)/A248(wt) and (pMini3bpUG and pMini3bpAG)/U248 combinations (ExpS 4.1 and 4.4) revealed a significant drop in kapp(+1) for A248(wt) by replacing U−1 with A−1, but only a two-fold rescue (cf. pMini3bpUG vs. pMini3bpAG) using U248. Interestingly, G248 cleaved the pMini3bpAG substrate at +1 with a markedly higher rate (kapp(+1)) compared to using the other 248 variants (Table 2; ExpS 4.1).
For the variants carrying "unnatural" nucleobases at −1 and/or at +73, most combinations resulted in cleavage mainly at +1 (Fig. 4D). The most apparent exceptions were for the combinations "pMini3bpDAPG/A248(wt)", "pMini3bp2APG/A248(wt)", and "pMini3bpUDAP/U248".
The pMini3bp variants cannot interact with the TBS-region (see above) and comparison of the pATSer (with T-loop) and the pMini3bp data sets (cf. Fig. 4B,D) revealed that some pATSer variants such as pATSerAG (ExpS 2.1), pATSerInoG (ExpS 2.7) and pATSerUDAP (ExpS 2.8; except U248) were cleaved at −1 with higher frequencies by all four 248 variants. Relative to cleavage of the pATSer derivatives with GAAA-tetra loops, the frequencies of cleavage at +1 were, in general, higher with the pMini3bp variants.
Considering rates of cleavage, introduction of 2NH2 [Table 2; cf. pMini3bpAG (ExpS 4.1) vs. pMini3bpDAPG (ExpS 4.8)] and removal of the 6NH2 [Table 2; cf. pMini3bpAG (ExpS 4.1) vs. pMini3bp2APG (ExpS 4.12)] on the −1 nucleobase resulted in a ≈four- and ≈ 160-fold decrease in kapp(+1) for G248, while for A248(wt) the corresponding values were ≈ three- and ≈ ten-fold lower. These data suggested that in particular the exocyclic amine at position 6 on A−1 plays a more important role for cleavage with G248 than for A248(wt). Cleavage of pMini3bpUG and pMini3bpUA with G248 resulted in a 20- and ten-fold lower kapp(+1), respectively, compared to A248(wt) (Table 2; ExpS 4.4 and 4.6) while only a small difference in kapp(+1) was detected for cleavage of pMini3bpUDAP using these two RPRs (Table 2; ExpS 4.11). Moreover, the kapp(+1) values for these three pMini3bp U−1 substrates using G248 were similar, within a factor of two. This might indicate that the catalytic performance of A248(wt) is influenced by pairing between N−1 and N+73 and/or the pairing between N+73 and U294 in the RPR-substrate complex.
In summary (see Fig. 5B), (i) the cleavage site distribution data did not provide any conclusive evidence for cis WC/WC pairing between residues N−1 and 248 in the Eco RPR substrate complex when we interfered with/or removed the interaction between the T-loop and TBS. However, there were a few possible exceptions, e.g., the combinations "pATSerCGGAAA/G248", "pATSerGGGAAA/C248", and "pMini3bpGG/C248". (ii) As in cleavage of pSu1 and pATSer variants, the potential pairing between N−1 and N+73 influence the efficiency of cleavage and site selection also in the absence of a productive interaction between the T-loop and TBS in the RPR. (iii) Interfering with the interaction between TSL and TBS affect choice of cleavage site and rate of cleavage, see also15,37.
Together the combined data with the four different substrate series suggested that the influence of N−1 and N+73 on cleavage site recognition and rate of cleavage at +1 and −1 depend on substrate and/or "N−1/N+73-N248" combination. Moreover, in general we do detect larger variations in kapp for cleavage at +1 than at −1. It therefore appears that the impact of the various changes either in the substrate or in the RPR is larger for cleavage at the correct position +1 than at −1. We also emphasize that the choice of cleavage site did not change during the course of the reactions as revealed from the time course experiments used to determine kapp values.
Altering the WC-surface of a U at position −1 and influence of the N248 identity
To further understand the importance of the Watson–Crick surface of the N−1 residue in the substrate we used substrates carrying substitutions of U with 3-methyl U (3mU) at −1. This modification would be expected to disturb the interaction with the Watson–Crick surface of U−1 (Fig. 3B–E). The data are shown in Fig. 6 and Table 3 (ExpS 5.1–5.3 vs. 2.4, 3.4 and 4.4).
A comparison of cleavage of pATSerUG vs. pATSer3mUG revealed no (or very minor) change in choice of cleavage site (Figs. 4B and 6; cf. ExpS 2.4 vs. ExpS 5.1) for any of the four 248 variants. However, kapp(+1) dropped three- to four-fold for all four RPRs, with U248 being the least efficient catalyst (Table 3).
With pATSerUGGAAA, introduction of 3mU at −1 did not result in any apparent change in cleavage site preference with the notable exception for U248. Here we did detect an increase of cleavage at +1 compared to cleavage of pATSerUGGAAA (Figs. 4C and 6; cf. ExpS 3.6 vs. ExpS 5.2). The other three 248 variants cleaved both pATSerUGGAAA and pATSer3mUGGAAA at +1. As for pATSerUG, the presence of 3mU−1 influenced cleavage rates; kapp(+1) values were down four- to six-fold using A248(wt) and G248, respectively, while for C248 the decrease was very modest, ≈1.5-fold. No apparent change was detected for U248. Interestingly, 3mU−1 influenced the rate of cleavage at +1 for A248(wt) and C248 while cleavage by G248 resulted in a four-fold decrease (Table 3; cf. ExpS 3.4 vs. ExpS 5.2).
Comparing cleavage of pMini3bpUG vs. pMini3bp3mUG, we detected just a small increase in cleavage at −1 for all 248 variants (Figs. 4D and 6; cf. ExpS 4.4 vs. ExpS 5.3). Moreover, kapp(+1) for A248(wt) was down 16-fold in response to the introduction of 3mU−1. For G248 and C248, the change was more modest, 2.7-fold lower for G248 while C248 cleaved 3mU−1 with a 2.5-fold higher rate than it cleaved the corresponding substrate lacking the methyl modification. No change was detected for U248.
In summary (see Fig. 5C), the presence of 3mU−1 that blocks the Watson–Crick surface has an impact on the rate of cleavage. The impact on the rate at +1 (kapp(+1)) appears to be dependent on RPR-substrate combination, as exemplified by cleavage of pMini3bpUG and pMini3bp3mUG with A248(wt) vs. C248. Remarkably, introduction of 3mU−1 in the "pATSer-GAAA-tetra-loop" substrate rescued cleavage at +1 using the U248 RPR variant. Hence, these findings do not support cis WC/WC pairing between N−1 and 248 for these substrates, see also29.
Kinetic constants kobs and kobs/Ksto and activation energy as a function of N248 identity
The data presented above clearly suggested that the identity of residue 248 affect both cleavage site recognition and rate of cleavage. We therefore decided to determine the kinetic constants, kobs and kobs/Ksto (for cleavage at +1), for the different 248 variants using pATSerUG. To gain insight into why a purine at 248 (in particular A at 248) is preferable over a pyrimidine, we also determined kobs and kobs/Ksto at different temperatures. This would allow us to estimate the activation energy for the reaction catalyzed by the various 248 RPRs. These series of experiments were done under single turnover conditions at 800 mM Mg2+ (see above) and the results are shown in Fig. 7 and Table 4.
Kinetics of cleavage of pATSer with the Eco RPR 248 variants and Arrhenius plots. (A) Rate of cleavage of pATSerUG as a function of increasing concentration of the Eco RPR 248 variants. The experiments were performed at 37 °C in buffer C containing 800 mM Mg2+ as described in “Materials and methods”. The data represent mean and experimental errors from at least three independent experiments. Insets correspond to Eadie–Hofstee plots using the primary data and the kobs and kobs/Ksto values presented in Table 4. (B) Arrhenius plots of temperature dependence of kobs for the Eco RPR248 variants as indicated. The data are summarized in Table 4 and the temperatures are in Kelvin. The values given in the inset correspond to the calculated Ea (activation energy) values.
The kobs and kobs/Ksto values for A248(wt) at 37 °C agreed with our previous data (Table 4)37. A comparison of kobs and kobs/Ksto for the four 248 variants revealed that having A or G at 248 resulted in the most efficient catalysts, in agreement with data discussed above. For A248(wt), lower temperature resulted in a modest but reproducible decrease in kobs. This trend was also detected for the other 248 variants. The kobs at different temperatures were highest for A248(wt) and G248, and lowest for C248 and U248. Irrespective of 248 variant and temperature, the Ksto values were similar within a ≈ two- to three-fold range. We have argued that under these reaction conditions Ksto ≈ Kd (see “Materials and methods”)31 and references therein. On the basis of this, our data suggested that substituting A248(wt) resulted in a modest change in binding affinity for pATSerUG.
Notwithstanding that the variation in kobs in response to temperature was modest (but reproducible) we plotted kobs as a function of temperature (Arrhenius plot). This would give an indication about the activation energy (Ea) for cleavage of pATSerUG by the different 248 variants. The Ea values varied from 12 to 57 kJ/mole, with A248(wt) having the lowest value followed by G248 < C248 and < U248 (Fig. 7; Table 4).
Taken together, in keeping with the data discussed above, a purine at 248 is preferred over a pyrimidine, with U248 being the weakest catalyst. From these data it also appears that this is, at least in part, due to the activation energy barrier being lower with a purine at 248, in particular with an adenosine as in Eco RPRA248(wt). This provides one rational why A at position 248 in bacterial RPR (Eco numbering) is conserved (see also the “Discussion”).
Differential effects due to replacement of the 2ʹOH at −1 with 2ʹH or 2ʹNH2 in pATSerUG and influence of the N248 identity on the charge distribution at the cleavage site
The 2ʹOH of residue −1 is important for both cleavage rates and site selection in bacterial RPR-mediated catalysis43. Hence, we decided to investigate whether replacement of the U−1 2ʹOH with 2ʹH or 2ʹNH2 in pATSerUG (pATSerdUG and pATSeramUG, respectively; Fig. 3B,E) influenced the choice of cleavage site.
Introduction of a 2ʹH (pATSerdUG) resulted in reduced cleavage at +1 for all 248 variants irrespective of pH (5.2, 6.1 and 7.2) consistent with previous data using pre-tRNA24,25. Importantly, cleavage at −1 did not increase with pH (Fig. 8A). Cleavage of the 2ʹNH2 substituted substrate (pATSeramUG) on the other hand resulted in increased cleavage at +1 at higher pH. In contrast to cleavage with A248(wt) and G248 higher pH was required to reach 50% cleavage at +1 using C248 (Fig. 8B). The most dramatic effect however, was observed using the U248 variant. Here we did not detect any significant change in the frequency of cleavage at +1 with increasing pH.
Frequencies of cleavage at +1 of different pATSerUG derivatives with 2ʹH or 2ʹNH2 at the −1 position at different pHs by Eco RPR 248 variants. (A) Histograms summarizing frequencies of cleavage at +1 in % during Eco RPR-mediated cleavage of pATSerdUG (2ʹOH at −1 substituted with 2ʹH). (B) Histograms summarizing frequencies of cleavage at +1 in % during Eco RPR-mediated cleavage of pATSeramUG (2ʹOH at −1 substituted with 2ʹNH2) and pATSeramUG(2AP+1/U+73). (C) Histograms summarizing frequencies of cleavage at +1 in % during Eco RPR-mediated cleavage of pATSeramUG(A+1/U+73) and pATSeramUG(2AP+1/U+73). (D) Histograms summarizing frequencies of cleavage at +1 in % during Eco RPR-mediated cleavage of pATSeramUG(DAP+1/U+73) and pATSeramUG(Ino+1/C+73). We used the 5ʹ cleavage fragments to calculate the frequencies of cleavage at +1 at different pH as indicated; mean and experimental errors were calculated from at least three independent experiments. For experimental details see “Materials and methods”.
The pH dependent cleavage of pATSeramUG at +1 by Eco RPRA248(wt) is also influenced by the identity of N+1/N+72 (cf. Fig. 5 in40; see also44; Fig. 8B,C; cf. G+1/C+72, A+1/U+72, 2AP+1/U+72, DAP+1/U+72 and Ino+1/C+72 substrate variants). This was also the case for the C248 and G248 variants. Of those substrate variants having an exocyclic amine at position 2 on the nucleobases (2NH2) at +1 (Fig. 3B; cf. substrates with G+1, 2AP+1 and DAP+1) C248 showed a similar response to pH as A248(wt), while higher pH was needed to reach 50% cleavage at +1 for G248 except using pATSeramUG(G+1/C+72) (cf. Fig. 8A–C). For the substrates lacking a 2NH2 on the nucleobase at +1 [pATSeramUG(A+1/U+72) and pATSeramUG(Ino+1/C+72)], we detected only a small increase in cleavage at +1 with increasing pH for A248(wt), C248 and G248 while for U248 no cleavage at +1 was observed. In fact, for U248 we observed no or only a small increase in cleavage at +1 using all pATSeramUG(N+1/N+72) variants with increasing pH. For all the RPR substrate combinations we also detected cleavage at other positions both downstream of the +1 site and in the 5ʹ leader with increasing pH (not shown). Also, irrespective of residue at 248 no significant change in the frequencies of cleavage at +1 with changing pH using the all ribo substrate variants was detected (not shown).
Taken together, these data suggest that the protonation (the pKa value) of the 2ʹNH2 at −1 is affected by the nucleobase identity at position 248 in Eco RPR and at +1 (and +72) in pATSerUG (see “Discussion”).
Discussion
Residues in the RNase P substrate interact with several regions of the RNA subunit (RPR) of bacterial RNase P (see introduction). Among these the N−1 residue in the substrate 5ʹ leader is close to the active center where cleavage occurs, and it has been proposed that the well conserved A248(wt) forms a cis WC/WC base pair when U is present at −124,25. These studies were primarily based on using pre-tRNAs carrying different deoxyribonucleobases at position N−1. In E. coli ≈40% of the pre-tRNAs do not carry a U at −124,27,28. Also, cross-linking studies suggest that N−1 and N+1 in the substrate are positioned close to A248-C253 and G332-A333 (E. coli numbering, see Fig. 1A)26,45,46. Hence, we have argued that A248(wt) is a key nucleobase of a N−1 binding surface/pocket16,27,29. Here we provide data where we analyzed cleavage as a function of A248(wt) substitutions and N−1 nucleobase identity using all ribo pre-tRNA and three all ribo model substrates to investigate whether N−1 and N248 forms a cis WC/WC base pair. If cis WC/WC base pair forms between N−1 and N248 this means that the phenotypic change due to disruption of the N−1/N248 pairing can be rescued by a compensatory change that restores pairing between N−1/N248. For the pre-tRNA substrate pSu1 and the model substrate pATSer, which both can form a productive TSL/TBS-interaction (see “Introduction”, induced fit mechanism)15,30,37,46, the data supported cis WC/WC pairing for substrates carrying G at −1, while we did not find any conclusive evidence for cis WC/WC pairing using the other combinations (except the U−1/A248 vs. A−1/U248 combinations in the pATSer context; see summary, Fig. 5A). When we interfered with the TSL/TBS-interaction by using "pATSer-GAAA-tetra-loop" substrates our findings are consistent with cis WC/WC pairing using the C−1, G−1 and U−1 substrate variants but not for A−1 (see summary, Fig. 5B). The impact of the N−1/N248 interaction was also detected using pre-tRNA substrates carrying a 2ʹH at −1 or substrates that could not form the RCCA-RNase P RNA interaction24,25, i.e. when additional RPR substrate interactions were disrupted. Moreover, our findings with the pMini3bp variants, which cannot interact with TBS in the S domain, lend less support for cis WC/WC pairing than when the "pATSer-GAAA-tetra-loop" series was used. But, support comes from using pMini3bpGG, pMini3bpDAPG and pMini3bpUDAP, where the latter can form three hydrogen bonds between N−1 and N+73 in the substrate (see summary, Fig. 5B). In summary, detection of possible cis WC/WC pairing between N−1 and N248 depends on substrate and disruption of more than one RPR-substrate contact such as the TSL/TBS-interaction.
Residue A248 is well conserved among bacterial RPRs and if the U−1 WC surface are involved in pairing with residue A248(wt) blocking the N3 position on the nucleobase—by adding a methyl group (3mU)—would interfere with choice of cleavage site and rate of cleavage. As in pSu1, the model substrates carry an A at −2. Hence, following Zahler et al.24,25, who used pre-tRNAAsp that also carries A−2, we argued that interfering with the formation of the "U−1/A248(wt)" potential pairing would result in a shift of cleavage from the correct site to the alternative site −1 due to the presence of the 3-methyl group at the N3 position of U−1 in the substrate. All three 3mU−1 model substrate variants were, however, preferentially cleaved at +1 irrespective of 248-variant. This is inconsistent with cis WC/WC pairing (see summary, Fig. 5C). Importantly, the introduction of 3mU−1 in the three all ribo model hairpin loop substrates did not shift choice of cleavage site for wild type Eco RPRA248(wt), which would be expected if there was cis WC/WC pairing between U−1 and A248(wt), see also29. It is also noteworthy that the presence of 3mU−1 in the "pATSer-GAAA-tetra-loop" substrate rescued cleavage at +1 using the U248 RPR variant. Together these data do not support cis WC/WC pairing between U−1 and A248(wt) in wild type Eco RPR. In this context we emphasize that substituting A248(wt) with U influenced the structure of the RPR, in particular in the P18 region, which has a role in connecting the S- and the C-domains. The P18 loop interacts with P8 and disruption of this interaction affects cleavage efficiency of both pre-tRNAs and model hairpin loop substrates47,48,49,50,51. Hence, this structural change in the RPR might therefore have an impact on the catalytic performance of the U248 variant, both with respect to site selection and rate of cleavage; however, again this would be substrate dependent. This would be in keeping with a perturbed coupling (i.e. induced fit, see e.g. Ref.15) between a productive TSL-TBS interaction and events at the cleavage.
Furthermore, in E. coli as well as in other bacteria a U is the most frequently (≈60%) occurring nucleobase at −1 in pre-tRNA 5ʹ leaders24,25,27,28. This also applies to the archaea Pyrococcus furiosus (65% U−1), which as E. coli possess a type A RPR and an A at the corresponding position to A2489,12,22 (Fig. 1B). As discussed above, there is limited support for cis WC/WC pairing between U−1 and A248(wt) in wild type Eco RPR. High GC-content bacteria such as Mycobacterium tuberculosis (and other mycobacteria; see Fig. 1B) and Neisseria meningitides carry type A RPRs with A248(wt) (E. coli numbering). In these bacteria, C at −1 is the most frequently occurring nucleobase, while U−1 is present in ≈13% and ≈32% of the pre-tRNAs, respectively27,28. This argues against formation of cis WC/WC pairing between N−1 and A248(wt) for the majority of pre-tRNAs in these bacteria.
In conclusion for the majority of pre-tRNAs (and model substrates), A248 does not interact with N−1 via cis WC/WC pairing. However, given that RNase P processes other RNA transcripts, including mRNAs2, we cannot completely exclude the possibility that A248(wt) is engaged in cis WC/WC pairing with these substrates. In this context we also have to consider that our experiments were performed without the C5 protein and hence the presence of C5 might have an impact given that C5 interact with residues upstream of N−1 (see above41,42). We propose that the structural architecture of the "active site" is flexible and varies dependent on the identity of the nucleobases at and near the cleavage site and their potential to interact with chemical groups in the RPR. This flexibility is also predicted to depend on the interaction between the pre-tRNA TSL-region and its binding site (TBS) in the RPR S-domain (see above) as well as the RCCA-RPR interaction15,24,25,30,37,44,46.
Structural architecture and Me(II)-binding near the cleavage site
RNase P mediated cleavage depends on Me(II)-ions, which are involved in activating the water molecule that acts as the nucleophile, substrate interaction and folding of the RPR43,52. On the basis of correctness and rate of cleavage available data suggest that Mg2+ is the preferred ion. Perreault and Altman53,54 suggested that binding of Mg2+ at the junction between the single stranded 5ʹ leader and the amino acid acceptor stem involves the two 2ʹ hydroxyls at positions −1 and −2 forming a productive complex that acts as the true RNase P substrate, see also25,38,39,46,55,56. In RNA the structural topology of Me(II)-binding sites affects both binding affinity and positioning of the Me(II)-ion. This is evident from lead(II)-induced cleavage studies of yeast tRNAPhe and Eco RPR15,57,58,59. For model substrates, introduction of U+1 (or C+1) in pATSerUG (or pATSerCG) affects lead(II)-induced cleavage at the cleavage site such that the frequency of cleavage 5ʹ of N+1 increases more than when a purine is present at +160. Similarly, substituting the 2ʹOH at -2, −1 and "C+74" in a model hairpin loop model substrate influences Mg2+-induced cleavage between −3 and −253. In keeping with this, substituting the N−1 2ʹOH with 2ʹNH2 in pATSerUG prevent Pb2+-induced cleavage between residue −1 and +1 (not shown). Also, the presence of a 2ʹNH2 at N−1 in pATSerUG (and pATSerCG) result in a shift of cleavage from −1 to +1 with increasing pH38,39,40,44; this report. The pKa for 2ʹNH2 is 6.0–6.2 (determined by NMR-spectroscopy using a dinucleotide)61,62. Therefore the 2ʹNH2 at −1 in pATSeramUG is most likely protonated at lower pH. As a consequence, this results in a positive charge at the +1 cleavage site, which interferes with cleavage at +1, causing the cleavage to shift to −138,39. With increasing pH, the 2ʹNH3+ becomes deprotonated, resulting in cleavage at +1. The pH dependent shift of cleavage from −1 to +1 (i.e., de-protonation of the 2ʹNH3+ at −1) is also dependent on the structure of the N+1/N+72 base pair40; this report. The data presented here using the 2ʹNH2 substituted substrates suggest that the identity of residue 248 in the RPR also influences the pH dependent shift from −1 to +1, in particular with respect to U248. However, we also observed a shift in the pH dependence for G248 when the structure of the N+1/N+72 base pair was altered. Given that A248(wt) is in close proximity to the cleavage site18 these data are consistent with a model where changes of the structural architecture at and near the cleavage site in the RPR-substrate complex (see above) affect the charge distribution. As a consequence, this influences the positioning of the Mg2+ that activates the water that acts as the nucleophile resulting in a shift of the phosphorus to be attacked31,43; for an alternative rational see25.
Proposed function of the well-conserved residue A248(wt) in wild type RPR and base stacking to prevent unspecific hydrolysis
In the RNase P tRNA crystal structure, which represents the post-cleavage stage, A248(wt) stacks on top of the tRNA G+1/C+72 base pair and presents the Hoogsteen surface facing the G+1 and the tRNA 5ʹ end (Fig. 9A)17; see also Refs20,63. The importance of the A248(wt) Hoogsteen surface for substrate interaction has been implicated on the basis of nucleotide analogue-modification interferences studies32. However, we provided data suggesting that the Hoogsteen surface of A248(wt) is not engaged in pairing with N−1, at least not in the case of pMini3bp substrates31. This raises the question about the role and function of A248(wt). The structure of yeast tRNAPhe reveals that the discriminator base at position +73 stacks on top of the G+1/C+72 pair (Fig. 9B)64. As such, the discriminator base acts as a hydrophobic cap that restricts access of bulk H2O to the terminal base pair65,66. Binding of pre-tRNA to the RPR results in formation of the RCCA-RNase P RNA interaction where the discriminator base pairs with residue U29418,27,34. In the RNase P-tRNA complex A248(wt) stacks on the G+1/C+72 base pair by occupying the position that the discriminator base has in free tRNA (Fig. 9A,B). This contributes to anchor the substrate to the RPR18,20,63. In addition, we propose that the A248(wt) stacking on G+1/C+72 prevents water from accessing the hydrophobic amino acid acceptor stem and potential unspecific hydrolysis of the tRNA after cleavage. We foresee that this also occurs prior to cleavage of the pre-tRNA and the recent cryoEM structures of Eco RNase P in complex with pre-tRNA support that this is indeed the case63. In this context the stacking free energy for A would be more favorable, followed by G, C and U67. Moreover, considering the activation energy (Ea), our findings indicated that the trend is A248(wt) < G248 < C248 < U248 with A248(wt) having the lowest activation energy barrier (Table 4). These data provide reasons to why A248(wt) in bacterial RPR is well conserved.
Illustration of base stacking. (A) Stacking of the discriminator base, D+73 (in magneta), on the G+1/C+72 base pair in the crystal structure of tRNAPhe (PDB code 1EVV)64. (B) Stacking of residue A248 (in magenta and E. coli numbering, Fig. 1) on the tRNAPhe G+1/C+72 base pair (in green) in the crystal structure of the RNase P-tRNAPhe complex (PDB code 3Q1R)18. Grey spheres represent Me(II)-ions. (C) Stacking and the RCCA-RPR interaction (in green) in the crystal structure of the RNase P-tRNAPhe complex (PDB code 3Q1R)18. Stacking residues in magenta. D73 corresponds to the discriminator base at position +73 in tRNA94 while the RPR numbering refers to E. coli numbering (Fig. 1). Note that A295 in E. coli corresponds to U266 in T. maritima RPR18. Stacking residues, the tRNA 3ʹ terminal A76 and the RPR residue, are marked in magenta. (D) Codon-anticodon interaction in the ribosomal A-site where residues in magenta stack as shown in the figure. p34–p37 correspond to positions in the tRNA anticodon loop. Gray residues represent the codon and residues marked in orange residues correspond to A1492 and A1493 in 16S rRNA (PDB code 2J02)80. (E,F) Stacking interactions in the ribosomal peptidyl transfer center, panel E (A-site) and panel F (P-site) as indicated. Orange residues correspond to rRNA residues interacting with the tRNA, green residues refer to tRNA and the tRNA discriminator base is highlighted in magenta (PDB code 5IBB)79. The images were created using PyMOL (Schrödinger, LLC).
We also note that the Ea value for cleavage of pATSerUG with A248(wt) was determined to be 12 kJ/mole (Table 4), which is two- to three-fold lower than for cleavage of pre-tRNATyrSu3, both with and without the RNase P protein C568. This difference could depend on substrate and/or reaction conditions. In pre-tRNATyrSu3 both the discriminator base (A+73) and the first 3ʹ C (C+74) pair with U−1 and G−2 in the 5ʹ leader, respectively, rendering A+73 and C+74 less accessible for interacting with RPR, i.e. formation of the "RCCA-RPR interaction" (see above13), compared to pATSerUG (Fig. 3). Also, here the experiments were performed at high Mg2+ and at a lower pH than in our previous study68, which are also factors to consider.
To conclude, in addition to its contribution to anchor the substrate18,20,63 we suggest that the function of A248(wt) is to replace the tRNA discriminator base and prevent access of water that would lead to unspecific hydrolysis/cleavage of the pre-tRNA in the RNase P-substrate complex. Saccharomyces cerevisiae RPR lacks an A at the position corresponding to Eco RPR A248(wt). Interestingly, in the cryo-EM structure of S. cerevisiae RNase P in complex with pre-tRNA the 5ʹ leader residues A−1 and A−2 stack on top of the tRNA G+1/C+72 pair forming a hydrophobic cap19. According to our proposal this would also prevent unspecific hydrolysis/cleavage of the pre-tRNA. Given that POP5 amino acid residues are also positioned close to the G+1/C+72 pair these might also contribute to prevent access of H2O and unspecific hydrolysis/cleavage (see also below).
Prevention of unspecific hydrolysis in PRORP
Like RNase P, proteinaceous PRORPs cleave the 5ʹ leader of pre-tRNAs and recent data show that the N−1 identity also influences cleavage by PRORPs both with respect to cleavage site recognition and rate of cleavage69,70,71. The crystal structures of PRORP1 and PRORP2 are available72,73; for a cryo structure see74, whereas the structure of PRORP in complex with its pre-tRNA substrate is not. Structural and mechanistic studies suggest that D474 and D475 coordinate Me(II) in the PRORP1 active site. Given the similarities between RNA and protein-based RNase P activities, i.e., the need to cleave pre-tRNAs correctly and prevent unspecific hydrolysis, it is likely that stacking on top of the N+1/N+72 base pair is also present in the PRORP-pre-tRNA complex. Candidates to act as a hydrophobic cap during the PRORP catalyzed reaction might be aromatic amino acids such as W478 and F500, which both are positioned close to the Me(II)-ion in the active site. Another possibility is that the pre-tRNA discriminator base keeps its position and stacks on top of the N+1/N+72 pair (and/or residues in the pre-tRNA 5ʹ leader, see above) in the PRORP-substrate complex as observed in other protein-tRNA complexes (see below). It will be interesting to determine whether this is the case and, if so, how access of water to the "inside" of the hydrophobic amino acid acceptor stem is prevented in the PRORP-substrate complex.
Base stacking and prevention of unspecific hydrolysis of RNA
Crystal structures of amino-acyl-tRNA synthetase-tRNA complexes (such as ArgRS-tRNAArg and MetRS-tRNAMet), EF-Tu-tRNAPhe, the CCA adding enzyme in complex with a tRNA mimic and tRNA bound to the ribosome show that the discriminator base at +73 stacks on the G+1/C+72 pair in a similar way as shown in Fig. 9A (see also E,F)75,76,77,78,79. In all these examples the discriminator is a purine. Moreover, inspection of the RCCA-RNase P RNA interaction in the RNase P-tRNA crystal structure reveals that U266 stacks on the A+73/U265 base pair, while the 3ʹ terminal A+76 stacks on the C+75/G263 base pair (Fig. 9C; note that the T. maritima residues G264, U265 and U266 correspond to G292, U294 and A295 in wild type Eco RPR, see Fig. 1)18. A similar type of stacking can also be observed in the ribosomal A- and P-sites both in the case of tRNA and mRNA interaction as well as with respect to the pairing between C74 and C75 and rRNA (Fig. 9D–F)79,80,81. Together this further emphasizes the importance of stacking. It is conceivable that a function of this "type" of base stacking is to prevent the access of water to functionally important base pairing interactions, and thereby ensuring high fidelity during RNA processing and decoding of mRNA.
Materials and methods
Preparation of substrates and RPR
The tRNASerSu1 precursor (pSu1) N−1 variants were generated as run-off transcripts using T7 DNA-dependent RNA polymerase and PCR-amplified templates as described elsewhere33,82. The model hairpin loop substrate N−1 series (pATSer, pATSer-GAAA-tetra loop and pMini3bp) were purchased from Thermo Scientific Dharmacon, USA. The substrates were [γ-32P]-ATP 5ʹ end-labeled and gel-purified followed by overnight Bio-Trap extraction (Schleicher and Schuell, GmbH, Germany; Elutrap in USA and Canada) and phenol–chloroform extraction as described elsewhere15,31.
The construction of the gene encoding Eco RPRG248 was recently reported31, while the C248 and U248 variants behind the T7 promoter were generated following the same procedure as outlined elsewhere using the wild type Eco RPRA248(wt) gene as template and appropriate oligonucleotides12,31,83,84. The RPRs were generated as run-off transcripts using T7 DNA-dependent RNA polymerase and PCR-amplified templates31,82.
Structural probing of the Eco RPR variants
The Eco RPR variants were 3ʹ-end labeled with [32P]pCp and structurally probed using Pb2+ and RNase T1 under native conditions as described elsewhere31,34,35,45,85. Briefly, approximately 2 pmols of labeled RPR in 10 µl was pre-incubated for 10 min at 37 °C in 50 mM Tris–HCl (pH 7.5), 100 mM NH4Cl and 10 mM MgCl2 together with 4 µM of the unlabeled corresponding RPR. Cleavage was initiated by adding freshly prepared Pb(OAc)2 to a final concentration of 0.5 mM and the reaction was stopped after 10 min. In the digestion with RNase T1, the RPR was pre-incubated as described above. One unit of RNase T1 was added followed by incubation on ice for 10 min. The reactions were stopped by adding two volumes of stop solution (10 M urea, 100 mM EDTA). The products were analyzed on 8% (w/v) denaturing polyacrylamide/7 M urea gels.
Cleavage assays and determination of kapp
The cleavage reactions were conducted in buffer C [50 mM 4-morpholineethanesulfonic acid (MES) and 0.8 M NH4Cl (pH 6.1)] at 37 °C and 800 mM Mg(OAc)2. The RPRs were pre-incubated at 37 °C in buffer C and 800 mM Mg(OAc)2 for at least 10 min to allow proper folding before mixing with pre-heated (37 °C) substrate. In all the experiments the concentrations of substrates were ≤ 0.02 µM, while the concentrations of the RPR variants were as indicated in Table and Figure legends. The reactions were terminated by adding two volumes of stop solution (see above). The products were separated on 25% (w/v) polyacrylamide/7 M urea gels.
Cleavage of pATSerUamG derivatives at 37 °C was performed in buffer C and 800 mM Mg(OAc)2 at pH 5.2, pH 6.1 and pH 7.239,40.
The rate constant kapp was determined under single-turnover condition at 800 mM Mg2+ in buffer C. The concentrations of Eco RPR variants used to generate the data are specified in the respective Table legends. The concentrations of pSu1 (precursor-tRNASerSu134) and model substrates15,31,34 were ≤ 0.02 μM (see also the main text). For rate calculations, we used the 5ʹ cleavage fragment as a measure of product formed. In each assay, the time of incubation was adjusted to ensure that the velocity measurements were in the linear range (typically ≤ 10%, but never exceeding that 40% of the substrate had been consumed). Each kapp value is reported as a mean ± deviation of this value, which was calculated using data (six time points) from at least three independent experiments.
Determination of the kinetic constants kobs, kobs/Ksto and Ksto
The rate constants kobs and kobs/Ksto were determined under saturating single-turnover conditions at pH 6.1 (where cleavage is suggested to be rate limiting) and 800 mM Mg2+ using pATSerUG, as described elsewhere, e.g.37. Under these conditions we have argued elsewhere that Ksto ≈ Kd in the Eco RPR-alone reaction12,30,31,86,87. The final concentrations of the different RPR variants were between 0.8 and 6.4 µM; the concentration of the pATSerUG substrate was ≤ 0.02 μM. To ensure that the experiments were done under single-turnover conditions, the lowest concentration of RPR was > 10 times higher than the concentration of the substrate. For the calculations we used the 5ʹ cleavage fragment, and the time of cleavage was adjusted to ensure that the velocity measurements were in the linear range (see above). To be able to compare with our previously published data, kobs and kobs/Ksto were obtained by linear regression from Eadie-Hofstee plots as described elsewhere12,30,31,37,88,89. Each value is an average of at least three independent experiments and is given as a mean ± the deviation of this value.
Data availability
The datasets used are available from the corresponding author.
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Acknowledgements
We thank our colleagues for discussions throughout this work and with respect to stacking special thanks to Dr J. Chattopadhyaya. Dr SG Svärd, Dr AC Forster and Ms T Bergfors for critically reading the manuscript.
Funding
Open access funding provided by Uppsala University. This work was funded by the Swedish Research Council (N/T), the Uppsala RNA Research Center (Swedish Research Council Linneus support) and Carl Tryggers Foundation to LAK. Funding for open access charge: Uppsala University.
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L.A.K. conceived the study. G.M., A.S.S., S.W. and D.K. performed the experiments and assisted in preparing the Figs. 1, 2, 3, 4, 5, 6, 7 and 8. L.A.K., G.M., A.S.S., S.W. and D.K. analyzed and interpreted the data. L.A.K. and G.M. wrote the manuscript. All authors read and approved the final version of the manuscript.
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Mao, G., Srivastava, A.S., Wu, S. et al. Importance of residue 248 in Escherichia coli RNase P RNA mediated cleavage. Sci Rep 13, 14140 (2023). https://doi.org/10.1038/s41598-023-41203-4
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DOI: https://doi.org/10.1038/s41598-023-41203-4
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