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The residue immediately upstream of the RNase P cleavage site is a positive determinant
Biochimie 84 (2002) 693–703
Original article
The residue immediately upstream of the RNase P cleavage site is a positive determinant Mathias Brännvall, B.M. Fredrik Pettersson, Leif A. Kirsebom * Department of Cell and Molecular Biology, Box 596, Biomedical Centre, 751 24 Uppsala, Sweden Received 6 May 2002; accepted 25 June 2002
Abstract We have studied the importance of the residue at the position immediately upstream of the RNase P RNA cleavage site using model substrates that mimic the structure at and near the cleavage site of the tRNAHis precursor. The various model substrates were studied with respect to cleavage site recognition as well as the kinetics of cleavage using M1 RNA, the catalytic subunit of Escherichia coli RNase P. Our studies showed that the identity of the residue immediately upstream of the cleavage site critically influences both these aspects. Among the ones tested, U is the preferred nucleotide at this position. Hence, these findings rationalize why most bacterial tRNAHis genes/transcripts harbor a U immediately upstream of the RNase P cleavage site and extend our understanding of the cleavage site recognition process in general and the unusual cleavage of the tRNAHis precursor in particular. Based on our as well as the data of others, we suggest that the nucleotide immediately upstream of the cleavage site is a positive determinant for cleavage by RNase P in general and the expression of tRNA genes is influenced by structural elements localized outside the promoter region i.e. in the leader and spacer regions of tRNA transcripts. © 2002 Éditions scientifiques et médicales Elsevier SAS. All rights reserved. Keywords: RNase P; Ribozyme; Divalent metal ions; TRNA precursors; TRNA processing
1. Introduction The tRNA genes are transcribed as precursors with extra nucleotides located both at the 5’- and 3’-termini as well as in the spacer regions between tRNA genes in multimeric tRNA transcripts. Consequently tRNA precursors have to be processed to generate functional tRNA (for a review see [1]). RNase P is an endoribonuclease responsible for generating the 5’ end of matured tRNA molecules. In Escherichia coli, this ribonucleoprotein complex consists of an RNA subunit and a basic protein, M1 RNA and C5, respectively. The catalytic activity is associated with the RNA, and correct and efficient cleavage occurs in vitro in the absence of protein and cleavage requires divalent metal ions, preferentially Mg2+ [2,3]. The residues at positions –2, –1, +1, +72, +73, +74 and +75 in a tRNA precursor are important for cleavage site recognition ([4] and references therein). The nucleotides at the 3’ end, the RCCA-motif, interact/base pair with RNase * Corresponding author. Tel.: +46-18-471-4068; fax: +46-18-53-03-96. E-mail address: [email protected] (L.A. Kirsebom). © 2002 Éditions scientifiques et médicales Elsevier SAS. All rights reserved. PII: S 0 3 0 0 - 9 0 8 4 ( 0 2 ) 0 1 4 6 2 - 1
P RNA; this is referred to as the “RNase P RNA-RCCA” interaction (interacting residues underlined; [5–8]). Moreover, we as well as others have provided evidence for the role of the residue at the –2 position in the substrate in the RNase P catalyzed reaction with respect to cleavage efficiency and cleavage site recognition [9–13]. In bacteria, the tRNAHis precursor is cleaved at the –1 position by RNase P generating a 5’ matured tRNA with an eight base pairs long amino acyl acceptor stem, here referred to as the aa-stem ([3] and references therein). This is in contrast to the other tRNA precursors that are cleaved at the canonical RNase P cleavage site, the +1 position, generating tRNA molecules with seven base pairs long aa-stems. Cleavage at –1 is in part due to the presence of discriminator base C73 in the tRNAHis precursor [14]. A C at this position in tRNA precursors interferes with cleavage by M1 RNA because the interaction between residues +73 in the substrate and U294 in M1 RNA is affected ([7,15]; see also below). In this report, we have investigated the importance of the residue at the position immediately upstream of the cleavage site using model substrates that mimic the structure at
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and near the cleavage site of the tRNAHis precursor. Note that the residue at the position immediately upstream of the cleavage site corresponds to –1 in “normal” tRNA precursors and –2 in the case of the tRNAHis precursor. Throughout this study, we will refer to this residue as residue –2. Our findings suggest that the residue at the –2 (–1 in normal tRNA precursors) position is a positive determinant in the M1 RNA catalyzed reaction. Our data raise the interesting possibility that other structural elements in tRNA genes localized outside the promoter region i.e. in the leader and spacer regions of tRNA transcripts, influence the expression level.
2. Materials and methods 2.1. Preparation of substrates and M1 RNA The various pATSer and pATHis derivatives were purchased from Xeragon AG, Switzerland or Dharmacon, USA and purified as described by Kufel and Kirsebom [16]. 5’ end-labeled substrates were generated and gel purified using standard protocols. The M1 RNA variants were generated as run-off transcripts using T7 DNA-dependent RNA polymerase [17] as described elsewhere [15,18]. 2.2. Assay conditions M1 RNA activity was monitored as previously described [2,19,20] in 50 mM Tris–HCl (pH 7.2), 5% (w/v) PEG 6000, 100 mM NH4Cl and 40 mM total divalent metal ions as indicated in the cleavage site recognition experiments, while 160 mM of Mg2+ were used in the kinetic experiments where the kinetic constants kcat and Km were determined. All the reactions were performed at 37 °C. 2.3. Determination of the M1 RNA cleavage site Determination of the sites of cleavage was performed essentially as described elsewhere [15] and inferred from the mobility of the 5’ cleavage products. The frequency of cleavage at different positions was quantified from the relative amounts of 5’ cleavage products generated from cleavage at the different positions using a PhosphoImager (Molecular Dynamics 400S). The concentrations of substrate and M1 RNA were 0.04 and 0.16 µM, respectively. 2.4. Determination of the kinetic constants kcat and Km The kinetic constants kcat and Km were determined as described in detail elsewhere [7,15,21]. The concentration of wild type M1 RNA was 0.041 µM except in the case of cleavage of pATHis“–2A” where the M1 RNA concentration was 0.082 µM. The concentrations of substrates varied between 0.08 and 421.6 µM depending on M1 RNA/substrate combination (cleavage of pATHis“–2A”,
substrate concentration 46–421.6 µM). The reaction products were separated on denaturing 20–22% (w/v) polyacrylamide gels and the amount of cleavage was quantified using a PhosphoImager (Molecular Dynamics 400S). For calculations, we used the 5’ cleavage fragments: the kcat and Km values were obtained by linear regression from Eadie– Hofstee plots and the ∆∆G values were calculated according to Wells [22].
3. Results In previous reports, we used a model substrate, pATSerCG (and derivatives thereof; Fig. 1), to study cleavage by M1 RNA in the presence of different divalent metal ions [19,21]. This substrate is derived from a tRNASer precursor as described elsewhere [15]. A derivative of pATSerCG, pATSerGC (here referred to as pATSerGC“–2A” where “–2A” denotes the identity of the residue at the –2 position), is cleaved preferentially at the –1 position by wild type M1 RNA while a mutant M1 RNA, M1G294 RNA (Fig. 2), cleaved this substrate mainly at +1 (Fig. 3 and Table 1). This is in keeping with our previous findings [15]. Thus, with respect to cleavage site recognition, cleavage of pATSerGC“–2A” by wild type M1 RNA is similar to cleavage of the tRNAHis precursor. However, cleavage of the tRNAHis precursor by the mutant M1G294 RNA resulted only in a low frequency of cleavage at the +1 position and only after cleavage had occurred at –1 suggesting the existence of other cleavage site recognition determinants in addition to the residue at +73 (which is part of the RCCARNase P RNA interaction; [7] and below). Comparison of the structural microenvironment at and near the cleavage site of pATSerGC“–2A” and the precursor to tRNAHis shows clear similarities with one striking difference; the former carries an A at –2 whereas the latter harbors a U at this position (Fig. 1). To explore this in detail and investigate the importance of the identity of residue –2 for M1 RNA mediated cleavage, we took advantage of this structural difference. We designed and generated a model substrate (and derivatives thereof) based on the tRNAHis precursor. This substrate, referred to as pATHis“–2U” (wild type), belongs to the same class of model substrates as pATSerGC“–2A” (see Fig. 1). 3.1. Residue –2 influences the cleavage site recognition The pATHis“–2U” (wild type) substrate, as pATSerGC“–2A”, was cleaved almost exclusively at the –1 position by wild type M1 RNA (Fig. 3 and Table 1). This result is expected given our previous data as well as the reports from other laboratories where the cleavage patterns of tRNAHis precursors were studied ([7] and references therein). Cleavage of pATHis“–2U” (wild type) by M1G294 RNA resulted only in a modest cleavage at +1 in keeping with the results using the tRNAHis precursor ([7]; see
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Fig. 1. The secondary structures of pATSer and pATHis derivatives are depicted. The substitutions that resulted in the various derivatives used in the present study are indicated. The arrows indicate the RNase P RNA cleavage sites.
above). However, these data are in contrast to the cleavage pattern observed for the other model substrate pATSerGC“–2A” that was cleaved preferentially at the +1 position by M1G294 RNA (Fig. 3 and Table 1). Thus, with respect to cleavage site recognition, these data suggest that pATHis“–2U” (wild type) can be considered as a model substrate that can be used to identify cleavage site recognition determinants in the tRNAHis precursor. In addition, these two model substrates behave differently when comparing cleavage by M1G294 RNA. The residues immediately upstream of the –1 cleavage site in pATHis“–2U” and pATSerGC“–2A” are different, U and A, respectively (Fig. 1). To investigate the contribution of the residue at –2 to cleavage site recognition (if any), we changed the A at –2 in pATSerGC“–2A” to a U (pATSerGC“–2U”) and studied the cleavage pattern of this variant for wild type M1 RNA and M1G294 RNA. The data are shown in Fig. 3 and summarized in Table 1. The cleavage patterns of pATSerGC“–2U” and pATHis“–2U” (wild type) were very similar and significantly different when compared to cleavage of pATSerGC“–2A” by these two M1 RNA variants. This suggests that residue –2 in pATSerGC plays a significant role for cleavage site recognition. This finding suggests that changing the corresponding residue in the pATHis“–2U” (wild type) substrate from a U to an A would influence cleavage site recognition such that cleavage of the pATHis“–2A” variant would be more similar to the cleav-
age pattern of pATSerGC“–2A”. As shown in Fig. 3 and Table 1, this is what we observed. However, comparing cleavage of pATHis“–2A” and pATSerGC“–2A” by the mutant M1G294 RNA still showed a clear difference with respect to preference of cleavage at –1 vs. the +1 position (Table 1). This finding suggests that pATHis“–2U” (wild type) harbors additional determinants involved in cleavage site recognition. Based on previous findings, this might involve the residue at the –3 position i.e. the nucleotide two bases upstream of the cleavage site (see [9]). We conclude that residue –2 in the model substrates used here function as a positive determinant (but not the sole determinant) for cleavage site recognition. 3.2. Addition of Sr2+ reduces cleavage at the –1 position We previously showed that cleavage in the presence of various combinations of divalent metal ions affects cleavage site recognition such that addition of transition metal ions increases miscleavage while cleavage in the presence of Sr2+ for example results in increased frequency of cleavage at the +1 position [15,21]. Here we wanted to investigate whether the cleavage pattern of the pATSerGC and pATHis derivatives changed upon addition of Sr2+ while keeping the total divalent metal ion concentration constant at 40 mM (see Section 2). The results are shown in Fig. 3 and are summarized in Table 1.
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Fig. 2. The secondary structure of E. coli RNase P RNA, M1 RNA, according to Haas and Brown [69]. The M1C294 RNA and M1G294 RNA derivatives carry changes at position 294 as indicated while the box indicated with dashed lines represents the “P15-loop”. The boxed “GGU-motif” is part of the RCCA-RNase P RNA interaction. For details see [15,21].
In the initial experiments, we observed that cleavage of pATSerGC“–2A” at –1 by wild type M1 RNA was reduced while cleavage at the +1 position appears to be unaffected (Fig. 3 and data not shown). This was also observed when U294 in M1 RNA was substituted with a C (Fig. 3 and Table 1; see also Fig. 2). Based on the results obtained for cleavage of the other model substrate derivatives, pATSerGC“–2U”, pATHis“–2U” and pATHis“–2A”, it appears that the Sr2+ induced decrease of cleavage at the –1 position depended on the identity of residue –2. By contrast, cleavage of pATSerCG at –1 by M1G294 RNA was not decreased upon addition of Sr2+ (data not shown; note that this substrate carries a G at +73 and a G at 294 in M1 RNA).
We also investigated the significance of the 2’ OH at position +73 in the substrate because previous data indicated participation of this 2’ OH in Mg2+ binding [23,24]. Substitution to deoxy at +73, pATSerGdC“–2A”, resulted in decreased cleavage at –1 upon addition of Sr2+ but to a lesser extent (Table 1). Additionally, the mutant M1G294 RNA cleaved pATSerGdC“–2A” at a higher frequency at –1 when compared to cleavage of pATSerGC“–2A” even in the presence of Mg2+ as the only divalent metal ion. Taken together, these data suggest that addition of Sr2+ influences cleavage site recognition and that the 2’ OH at position +73 plays a role in cleavage site recognition. In addition, it appears that the identities of residues +73 and –2 in the
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Fig. 3. 5’-cleavage fragments generated after cleavage of various [γ-32P]-ATP 5’ end-labeled model substrates by different M1 RNA derivatives. The experiments were performed as described in Section 2 at 40 mM final concentration of divalent metal ions. In the experiments where Mg2+ and Sr2+ were mixed, the final concentration of each was 20 mM. The time of incubation of M1 RNA in the presence of the various model substrates is indicated. (A) Cleavage of pATHis derivatives and (B) cleavage of pATSer derivatives. The controls consisted in incubating model substrates in the presence 20 mM Mg2+ and 20 mM Sr2+ and no M1 RNA: (I) pATHis“–2U” (wild type); (II) pATHis“–2A”; (III) pATSerGdC“–2A”; (IV) pATSerGC“–2A” and (V) pATSerGC“–2U”.
substrate and 294 in M1 RNA affect the Sr2+-induced decrease of cleavage at –1. 3.3. Residue –2 influences the kinetics of cleavage at –1 Next we determined the kinetic constants for cleavage of the various substrates described above except pATSerGdC“–2A”. In our initial experiments at [Mg2+] = 40 mM where we studied cleavage by wild type M1 RNA, we were unable to reach substrate saturation when we plotted initial rates as a function of substrate
concentration. (Note the absence of the +73/294 interaction in the RS-complex for the different M1 RNA substrate combinations used in the present study, see Fig. 4.) We therefore decided to increase the Mg2+-concentration to 160 mM since it has previously been shown that an increase in Mg2+ concentration influences the kinetic constants in cleavage of model substrates [24]. At 160 mM, Mg2+ substrate saturation was observed for the various M1 RNA substrate combinations studied indicating that an increase in Mg2+ can stabilize the interaction between M1 RNA and the model substrates used in this report (see also below). As a
Table 1 Summary of frequencies of cleavage of the various substrates used in the present study. The frequencies of cleavage at –1 and at +1 are given in percentage (± experimental errors only given for cleavage at –1) and are averages of several independent experiments. The experiments were performed in the presence of 40 mM Mg2+ as described in Section 2. Note that for the tRNAHis precursor the –1 position is the natural RNase P cleavage site. Substrate
pATSerCG“–2A” pATSerGC“–2A”
pATSerGdC“–2A” pATSerGC“–2U” pATHisGC“–2U” pATHisGC“–2A” . a
M1 RNA variant identity of residue at position 294 U294 (wild type) G294 a U294 C294 G294 U294 G294 U294 G294 U294 G294 U294 G294
Numbers taken from [15].
a
Cleavage site [Mg2+] = 40 mM
Cleavage site [Mg2+] = 20 mM; [Sr2+] = 20 mM
–1
+1
–1
+1
2.4 84 90 ± 2.7 74 ± 8.4 14 ± 4.5 97 ± 0.5 40 ± 1 >99 94 ± 0.5 99 ± 0.2 99 ± 0.3 94 ± 1 75 ± 2
97.6 16 10 26 86 3 60 <1 6 1 1 6 25
55 ± 3.9 40 ± 7.2 9.5 ± 3.4 86 ± 4 21 ± 2 95 ± 2 82 ± 1 >99 98 ± 0.5 78 ± 5 56 ± 2
45 60 90.5 14 79 5 18 <1 2 22 44
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Fig. 4. Model of RNase P RNA mediated cleavage of RNA. Top, simplified scheme of the cleavage reaction, see also [15]. Bottom, representation of RS-complexes that result in cleavage preferentially at +1 (RS’’) and at –1 (RS’), respectively. The model suggests that cleavage at +1 is preceded by two intermediates, RS’ and RS’’, and cleavage at +1 is dictated by the rate constants kchem–1 and k+2 (k–2), for details see text. In this model, a pyrimidine at –2 (or –1 when cleavage occurs at +1; boxed residues, RS’’ in A and RS’ in B, respectively) influences the rate of cleavage at the respective position by mediating a positive interaction with M1 RNA (for details see text). Three divalent metal ions are depicted in the figure, 1–3; two (2) and (3) have been localized in the P15-loop [16,35] and one (1) is positioned in the vicinity of the cleavage site [21,24,30,35]. Small arrows indicate the cleavage sites. Note that cleavage at the –1 position also occurs in the presence of the +73/294 interaction and that frequency of cleavage at –1 increases with increasing concentration of Mg2+ (Table 2) or as the result of addition of transition metals [15,21].
reference substrate, we used pATSerCG that is the “wild type” pATSer derivative [15,21]. The data are summarized in Table 2. As a result of increasing the concentration of Mg2+ we observed a threefold stimulation of the efficiency of cleavage of the reference substrate, pATSerCG, which mainly was due to a decrease in Km indicating an improvement in the interaction between the substrate and M1 RNA. In addition, an increased miscleavage at the –1 position was observed. The pATSerGC“–2A” substrate that is cleaved mainly at –1 was cleaved with a significant increase in Km (compared to cleavage of pATSerCG at –1) which corresponds to a loss of ≈1.7 kcal/mol. This was expected and is in keeping with an impaired interaction between C+73 in the
substrate and U294 in M1 RNA (see Fig. 4 and below). The wild type pATHis“–2U” substrate was cleaved approximately threefold more efficiently when compared to pATSerGC“–2A” with significant differences in both kcat and Km. Thus, the kinetics of cleavage differed when comparing cleavage of these two substrates. Substitution of residue –2 in pATSerGC“–2A” to a U (pATSerGC“–2U”) resulted in an almost 200-fold increase in kcat, while the U to A replacement in pATHis“–2A” resulted in 10- and threefold changes in kcat and Km, respectively. This discrepancy, kcat effect for pATSer vs. kcat and Km effect for pATHis, might be due to the presence of additional determinants for cleavage site recognition for cleavage of pATHis (see above) that also affect the kinetics of cleavage.
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Table 2 Summary of the kinetic constants, kcat and Km, in cleavage of the various model substrates by wild type M1 RNA. ∆∆G (kcal × mol–1)
Substrate
Residue “–2” in the substrate
M1 RNA pos 294
Cleavage site
kcat (min–1)
Km (µM)
kcat/Km (µM–1 × min–1)
pATSerCG a [Mg2+] = 40 mM
A
U
–1 +1
0.13 ± 0.043 4.2 ± 2.4
4.3 ± 2.4 4.3 ± 2.4
0.027 0.97
pATSerCG [Mg2+] = 160 mM pATSerGC“–2A” [Mg2+] = 160 mM pATSerGC“–2U” [Mg2+] = 160 mM pATHisGC“–2U” [Mg2+] = 160 mM pATHisGC“–2A” [Mg2+] = 160 mM .
A
U
A
U
U
U
–1 +1 –1 +1 –1
0.08 ± 0.05 1.2 ± 0.78 0.07 ± 0.04 0.007 b 13 ± 2.3
0.4 ± 0.15 0.4 ± 0.15 3.6 ± 1.6 3.6 b 4.1 ± 0.6
0.2 3.1 0.019 0.0019 b 3.2
–3.2 3
U
U
–1
4.7 ± 0.7
72 ± 7
0.065
–2.3 4
A
U
–1
0.4 ± 0.2
266 ± 120
0.0015
14
13
(1) 1 (1) 2 (+1.4) 1 (+4.6) 2
Each value is an average of several independent experiments and kcat and Km values were determined as outlined in Section 2. Superscripts (1 to 4) in the ∆∆G column refer to the ∆∆G values that were compared. a Numbers taken from Brännvall and Kirsebom, 2001 [21]. ∆∆G values were calculated according to Wells [22] and ∆∆G values given in parenthesis were calculated comparing cleavage of pATSerCG (wild type) and pATSerGC(“–2A”). b Calculation of kcat/Km for cleavage at +1 was performed based on the fact that the cleavage frequency at +1 = 10% (Table 1) and our previous findings showing that the Km is approximately the same irrespective of cleavage site [15,21].
Nevertheless, calculation of ∆∆G values reveals a significant loss of energy as a result of replacement of the –2U with an adenosine in both substrates (Table 2). Taken together, these findings demonstrate that residue –2 plays a significant role with respect both to cleavage site recognition and kinetics.
4. Discussion The data reported here extend our understanding of the cleavage site recognition process in general and the unusual cleavage of the tRNAHis precursor in particular. The unusual cleavage of the tRNAHis precursor at the –1 position has previously been shown to be due to the primary structure of the aa-stem with the G–1C+73 base pair of particular importance [7,20,25–27]. Based on the available tRNA gene sequences, it was noted that E. coli tRNA genes/transcripts preferentially carry a uridine at the position immediately upstream of the canonical RNase P cleavage site i.e. at the –1 position (–2 for tRNAHis) [8,28]. (In fact comparison of bacterial tRNA gene sequences that are available today reveals that in most bacteria the preferred nucleotide at the –1 position is a uridine (unpublished).) As shown in Table 3, where we compared the primary structures of tRNAHis genes from various bacteria and three Archaea, all, with one exception, harbor a U at –2 i.e. position immediately upstream of the RNase P cleavage site. This is rationalized by our present findings that clearly demonstrates that both the kinetics of cleavage and cleavage site recognition of the two model substrates used in the present report are influenced by the identity of residue –2 where a uridine is the preferred nucleotide among the ones tested.
4.1. Possible role(s) of residue –2 Replacement of the C–1G+73 base pair to a G–1C+73 in the model substrate pATSerCG resulted in substantial cleavage at –1. This miscleavage was reversed by changing U294 in M1 RNA to a G ([15]; this report). Substitution of C+73 in the tRNAHis precursor or cleavage of the wild type tRNAHis precursor by M1G294 RNA resulted in cleavage both at –1 and at +1 [7,25,26]. Mutational analysis of the +73/294 interaction showed that the kinetics of cleavage changed such that the rate of product release is increased in the absence of the interaction [7,20]. These data clearly suggest that residue +73 in the substrate interacts with 294 in M1 RNA and that this interaction is important for cleavage site recognition and the kinetics of cleavage. In cleavage of the model pATSer substrate, the absence of this interaction resulted in a loss of 1.4 and 4.6 kcal/mol for cleavage at –1 and at +1, respectively, corresponding roughly to a base pair. Additionally, our recent data suggest that the interaction between +73 and 294 is stabilized by Mg2+ [21]. Moreover, in almost half of the tRNA precursors in E. coli, the residue at –1 is paired with the base at position +73 i.e. the discriminator position. Therefore, it is most likely that the residue at –1 interacts with M1 RNA as a result of formation of the +73/294 interaction in the RS-complex as discussed elsewhere [9]. This would expose the cleavage site as we have suggested previously [5]. We recently suggested that cleavage by M1 RNA proceeds via intermediates according to the simplified scheme depicted in Fig. 4 [15] . In this model, cleavage at –1 is dictated by the rate constants kchem–1 and k+2 (and k–2). The former represents the constant for the chemistry of cleavage while the latter is the rate constant for breaking the –1/+73
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Table 3 Distribution of bases at the RNase P cleavage site on the tRNAHis precursor. Organism Bacillus subtilis 168 Bacillus subtilis 168 Borrelia burgdorferi B31 Campylobacter jejuni NCTC 11168 Clostridium perfringens 13 Escherichia coli K12-MG1655 Haemofilus influenczae Rd (KW20) Listeria monocytogenes EGD-e Mycobacterium leprae TN Mycobacterium tuberculosis H37Rv Mycoplasma genitalium G-37 Mycoplasma pneumoniae M129 Neisseria meningitideis MC58 Pseudomonas aeruginosa PA01 Rickettsia prowazekii Madrid E Salmonella typhimurium LT2 Staphylococcus aureus Mu50 Streptomyces coelicolor A3(2) Thermatoga maritima MSB8 Treponema palladiium Nichols Vibrio cholerae El Tor N16961 Vibrio cholerae El Tor N16961 Yersinia pestis CO92 Archaeoglobus fulgidus DSM4304 Methanococcus jannaschii DSM2661 Sulfolobus solfataricus P2 . a b
tRNA a
trnB trnD a
b
b
b
His1 His2
Position –2
Position –1
Reference
U U U U U U U U U U U U U U U U U U U C U U U U U U
G G G G G G G G G G G G G G G G G G G G G G G G G G
[46] [46] [45] [67] [68] [47] [55] [63] [49] [51] [48] [50] [56] [52] [53] [58] [57] [62] [59] [60] [54] [54] [61] [64] [65] [66]
Both tRNAs are identical except for a C to U change in the variable loop. Harbors two genes annotated as tRNAHis genes.
base pair in the substrate and forming the +73/294 interaction in the RS-complex. Thus, a slow k+2 (due to the absence of the +73/294 interaction) will favor cleavage at the –1 position as in cleavage of pATSerGC“–2A” by wild type M1 RNA. By contrast, when k+2 is fast i.e. the +73/294 interaction is established (going from RS’ to RS’’; Fig. 4) as in the case of cleavage of pATSerGC“–2A” with M1G294 RNA, then cleavage will preferentially occur at the +1 position. Replacement of the –2A with a –2U in pATSerGC“–2A” resulted in a significant increase in kcat (Table 2). Product release is apparently not the rate-limiting step in cleavage of this type of model substrates [24]. Hence, the given kcat values most likely describe the reaction prior to release of the product(s). The increase in kcat also indicates a role for residue –2 going from the ground state to the transition state. This is associated with a change in ∆∆G of 3.2 kcal/mol (–2A compared to –2U; Table 2), which roughly corresponds to formation/disruption of a base pair interaction. Thus, a likely possibility is that a U at the –2 position affects the rate of cleavage by mediating a positive interaction with M1 RNA such that kcat is increased (Fig. 4). In this context, we have previously discussed the possibility that a U at –1 (which corresponds to –2 in tRNAHis) might interact with the J18/2 region (A328–U335) ([8]; see also [29]). However, the J5/15 (A248, A249) and P15 (C252, C253) regions must also be considered since cross-linking data suggest that residues in these regions are in close proximity to residue –1 [8]. Alternatively, but not mutually
exclusive, a U immediately upstream of the cleavage site might affect coordination of (a) Mg2+-ion(s) important for the chemistry of cleavage. In fact it is clear that Mg2+ binds in the vicinity of the RNase P cleavage site [21,23,24,30]. In this context, we note that the “wild type” pATSerCG substrate was miscleaved at a higher frequency at elevated Mg2+ concentrations (Table 1 and data not shown). At present we cannot distinguish between these possibilities or exclude other explanations. For example we note that for in vitro modified yeast tRNAPhe precursors the 2’ OH immediately upstream of the canonical RNase P cleavage site was suggested to mediate an interaction with RNase P RNA that plays a role for positioning of the scissile bond in the active site [12]. Together with our findings, this scenario would suggest that the identity of residue –2 influences formation of the (–2) “2’ OH–M1 RNA” interaction and/or positioning of the scissile bond. Clearly, more detailed experiments have to be performed to establish the function of the nucleotide immediately upstream of the cleavage site in the RNase P RNA catalyzed reaction. 4.2. Importance of the 2’ OH at position +73 in the substrate Several 2’ OH in the substrate have been implicated to play a role in the interaction with RNase P RNA and in coordination with Mg2+ [8,23,24,31–35]. Among these 2’ OH groups, the 2’ OH at the +73 position has been suggested to be involved in coordination of Mg2+ [23,36].
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Here we showed that removal of the 2’ OH at +73 resulted in an increased frequency of cleavage at the –1 position when compared to cleavage in its presence (Table 2; compare cleavage of pATSerGC“–2A” and pATSerGdC“–2A” with M1G294 RNA). This is in keeping with data where we studied cleavage of other pATSer derivatives by wild type M1 RNA (to be published elsewhere). Moreover, addition of Sr2+ appeared to result in a reduction of cleavage of pATSerGC“–2A” by wild type M1 RNA preferentially at –1. In the absence of the 2’ OH at +73, the Sr2+-induced decrease of cleavage at –1 was less accentuated (Table 2). A structural study of a model of tRNAAla showed that Sr2+ coordinates to 2’ OH groups [37]. It is therefore likely that Sr2+ utilize the 2’ OH at +73 as a ligand and thereby displace Mg2+. As a consequence, this results in a reduced rate of cleavage at –1. Taken together, our present findings lend support to the involvement of the 2’ OH at +73 in Mg2+ binding and suggest that this Mg2+ influence cleavage site recognition. In view of our recent findings [21], this Mg2+ is likely to be involved in metal ion cooperativity in cleavage by RNase P RNA. 4.3. Expression of tRNA genes is influenced by structural elements localized outside the promoter region Substitution of the nucleotide at the +2 position in tRNATyrSu3 from a G to an A results in a reduction of the intracellular concentration of 5’ matured tRNA. The reduction appears to be complemented by a C to U change at position –4 in the leader [38]. This finding might be rationalized in view of more recent data that suggest that the RNase P protein is positioned close to the cleavage site and enhances the interaction with the 5’ leader ([39]; see also [29,40]). Thus, it is possible that substitution of residue –4 influences this interaction and thereby RNase P cleavage efficiency. Moreover, in vitro studies with Bacillus subtilis RNase P demonstrate that the length of the 5’ leader influenced the catalytic efficiency ([41]; see also [42]). Krupp et al. [42] also noted that cleavage between pyrimidine–purine bonds was preferred relative to cleavage of purine–purine bonds. Recently it was demonstrated that a C to A (or G) change in a model substrate reduced cleavage for the RNase P holoenzyme derived from E. coli as well as B. subtilis [43] consistent with our present findings (Table 2). These in vitro studies are in keeping with our tRNATyrSu3 nonsense suppressor data where replacement of –1U to –1A resulted in a significant reduction in suppression efficiency [44] as well as the low appearance of a purine at the –1 position in tRNA genes/transcripts. Finally, cleavage of various tRNA-like structures with RNase P RNA in the presence/absence of the RNase P protein suggests that the nucleotide at the position corresponding to the –1 position in a tRNA precursor plays an important role in the reaction [28]. In conclusion, we suggest that the nucleotide immediately upstream of the cleavage site is a positive determinant
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for cleavage by RNase P. In addition, expression of tRNA genes is influenced by structural elements localized outside the promoter region i.e. in the leader and spacer regions of tRNA transcripts. Based on this and the data reported here raise the possibility that a U to A change at –2 in the tRNAHis gene in for example E. coli might lower expression of tRNAHis.
Acknowledgements We thank our colleagues for discussions, Dr. S. Dasgupta for critical reading of the manuscript. This work was supported by grants from the Swedish Natural Science Research Council, the Foundation for Strategic Research and the Wallenberg Foundation (to L.A.K.).
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Original article
The residue immediately upstream of the RNase P cleavage site is a positive determinant Mathias Brännvall, B.M. Fredrik Pettersson, Leif A. Kirsebom * Department of Cell and Molecular Biology, Box 596, Biomedical Centre, 751 24 Uppsala, Sweden Received 6 May 2002; accepted 25 June 2002
Abstract We have studied the importance of the residue at the position immediately upstream of the RNase P RNA cleavage site using model substrates that mimic the structure at and near the cleavage site of the tRNAHis precursor. The various model substrates were studied with respect to cleavage site recognition as well as the kinetics of cleavage using M1 RNA, the catalytic subunit of Escherichia coli RNase P. Our studies showed that the identity of the residue immediately upstream of the cleavage site critically influences both these aspects. Among the ones tested, U is the preferred nucleotide at this position. Hence, these findings rationalize why most bacterial tRNAHis genes/transcripts harbor a U immediately upstream of the RNase P cleavage site and extend our understanding of the cleavage site recognition process in general and the unusual cleavage of the tRNAHis precursor in particular. Based on our as well as the data of others, we suggest that the nucleotide immediately upstream of the cleavage site is a positive determinant for cleavage by RNase P in general and the expression of tRNA genes is influenced by structural elements localized outside the promoter region i.e. in the leader and spacer regions of tRNA transcripts. © 2002 Éditions scientifiques et médicales Elsevier SAS. All rights reserved. Keywords: RNase P; Ribozyme; Divalent metal ions; TRNA precursors; TRNA processing
1. Introduction The tRNA genes are transcribed as precursors with extra nucleotides located both at the 5’- and 3’-termini as well as in the spacer regions between tRNA genes in multimeric tRNA transcripts. Consequently tRNA precursors have to be processed to generate functional tRNA (for a review see [1]). RNase P is an endoribonuclease responsible for generating the 5’ end of matured tRNA molecules. In Escherichia coli, this ribonucleoprotein complex consists of an RNA subunit and a basic protein, M1 RNA and C5, respectively. The catalytic activity is associated with the RNA, and correct and efficient cleavage occurs in vitro in the absence of protein and cleavage requires divalent metal ions, preferentially Mg2+ [2,3]. The residues at positions –2, –1, +1, +72, +73, +74 and +75 in a tRNA precursor are important for cleavage site recognition ([4] and references therein). The nucleotides at the 3’ end, the RCCA-motif, interact/base pair with RNase * Corresponding author. Tel.: +46-18-471-4068; fax: +46-18-53-03-96. E-mail address: [email protected] (L.A. Kirsebom). © 2002 Éditions scientifiques et médicales Elsevier SAS. All rights reserved. PII: S 0 3 0 0 - 9 0 8 4 ( 0 2 ) 0 1 4 6 2 - 1
P RNA; this is referred to as the “RNase P RNA-RCCA” interaction (interacting residues underlined; [5–8]). Moreover, we as well as others have provided evidence for the role of the residue at the –2 position in the substrate in the RNase P catalyzed reaction with respect to cleavage efficiency and cleavage site recognition [9–13]. In bacteria, the tRNAHis precursor is cleaved at the –1 position by RNase P generating a 5’ matured tRNA with an eight base pairs long amino acyl acceptor stem, here referred to as the aa-stem ([3] and references therein). This is in contrast to the other tRNA precursors that are cleaved at the canonical RNase P cleavage site, the +1 position, generating tRNA molecules with seven base pairs long aa-stems. Cleavage at –1 is in part due to the presence of discriminator base C73 in the tRNAHis precursor [14]. A C at this position in tRNA precursors interferes with cleavage by M1 RNA because the interaction between residues +73 in the substrate and U294 in M1 RNA is affected ([7,15]; see also below). In this report, we have investigated the importance of the residue at the position immediately upstream of the cleavage site using model substrates that mimic the structure at
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and near the cleavage site of the tRNAHis precursor. Note that the residue at the position immediately upstream of the cleavage site corresponds to –1 in “normal” tRNA precursors and –2 in the case of the tRNAHis precursor. Throughout this study, we will refer to this residue as residue –2. Our findings suggest that the residue at the –2 (–1 in normal tRNA precursors) position is a positive determinant in the M1 RNA catalyzed reaction. Our data raise the interesting possibility that other structural elements in tRNA genes localized outside the promoter region i.e. in the leader and spacer regions of tRNA transcripts, influence the expression level.
2. Materials and methods 2.1. Preparation of substrates and M1 RNA The various pATSer and pATHis derivatives were purchased from Xeragon AG, Switzerland or Dharmacon, USA and purified as described by Kufel and Kirsebom [16]. 5’ end-labeled substrates were generated and gel purified using standard protocols. The M1 RNA variants were generated as run-off transcripts using T7 DNA-dependent RNA polymerase [17] as described elsewhere [15,18]. 2.2. Assay conditions M1 RNA activity was monitored as previously described [2,19,20] in 50 mM Tris–HCl (pH 7.2), 5% (w/v) PEG 6000, 100 mM NH4Cl and 40 mM total divalent metal ions as indicated in the cleavage site recognition experiments, while 160 mM of Mg2+ were used in the kinetic experiments where the kinetic constants kcat and Km were determined. All the reactions were performed at 37 °C. 2.3. Determination of the M1 RNA cleavage site Determination of the sites of cleavage was performed essentially as described elsewhere [15] and inferred from the mobility of the 5’ cleavage products. The frequency of cleavage at different positions was quantified from the relative amounts of 5’ cleavage products generated from cleavage at the different positions using a PhosphoImager (Molecular Dynamics 400S). The concentrations of substrate and M1 RNA were 0.04 and 0.16 µM, respectively. 2.4. Determination of the kinetic constants kcat and Km The kinetic constants kcat and Km were determined as described in detail elsewhere [7,15,21]. The concentration of wild type M1 RNA was 0.041 µM except in the case of cleavage of pATHis“–2A” where the M1 RNA concentration was 0.082 µM. The concentrations of substrates varied between 0.08 and 421.6 µM depending on M1 RNA/substrate combination (cleavage of pATHis“–2A”,
substrate concentration 46–421.6 µM). The reaction products were separated on denaturing 20–22% (w/v) polyacrylamide gels and the amount of cleavage was quantified using a PhosphoImager (Molecular Dynamics 400S). For calculations, we used the 5’ cleavage fragments: the kcat and Km values were obtained by linear regression from Eadie– Hofstee plots and the ∆∆G values were calculated according to Wells [22].
3. Results In previous reports, we used a model substrate, pATSerCG (and derivatives thereof; Fig. 1), to study cleavage by M1 RNA in the presence of different divalent metal ions [19,21]. This substrate is derived from a tRNASer precursor as described elsewhere [15]. A derivative of pATSerCG, pATSerGC (here referred to as pATSerGC“–2A” where “–2A” denotes the identity of the residue at the –2 position), is cleaved preferentially at the –1 position by wild type M1 RNA while a mutant M1 RNA, M1G294 RNA (Fig. 2), cleaved this substrate mainly at +1 (Fig. 3 and Table 1). This is in keeping with our previous findings [15]. Thus, with respect to cleavage site recognition, cleavage of pATSerGC“–2A” by wild type M1 RNA is similar to cleavage of the tRNAHis precursor. However, cleavage of the tRNAHis precursor by the mutant M1G294 RNA resulted only in a low frequency of cleavage at the +1 position and only after cleavage had occurred at –1 suggesting the existence of other cleavage site recognition determinants in addition to the residue at +73 (which is part of the RCCARNase P RNA interaction; [7] and below). Comparison of the structural microenvironment at and near the cleavage site of pATSerGC“–2A” and the precursor to tRNAHis shows clear similarities with one striking difference; the former carries an A at –2 whereas the latter harbors a U at this position (Fig. 1). To explore this in detail and investigate the importance of the identity of residue –2 for M1 RNA mediated cleavage, we took advantage of this structural difference. We designed and generated a model substrate (and derivatives thereof) based on the tRNAHis precursor. This substrate, referred to as pATHis“–2U” (wild type), belongs to the same class of model substrates as pATSerGC“–2A” (see Fig. 1). 3.1. Residue –2 influences the cleavage site recognition The pATHis“–2U” (wild type) substrate, as pATSerGC“–2A”, was cleaved almost exclusively at the –1 position by wild type M1 RNA (Fig. 3 and Table 1). This result is expected given our previous data as well as the reports from other laboratories where the cleavage patterns of tRNAHis precursors were studied ([7] and references therein). Cleavage of pATHis“–2U” (wild type) by M1G294 RNA resulted only in a modest cleavage at +1 in keeping with the results using the tRNAHis precursor ([7]; see
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Fig. 1. The secondary structures of pATSer and pATHis derivatives are depicted. The substitutions that resulted in the various derivatives used in the present study are indicated. The arrows indicate the RNase P RNA cleavage sites.
above). However, these data are in contrast to the cleavage pattern observed for the other model substrate pATSerGC“–2A” that was cleaved preferentially at the +1 position by M1G294 RNA (Fig. 3 and Table 1). Thus, with respect to cleavage site recognition, these data suggest that pATHis“–2U” (wild type) can be considered as a model substrate that can be used to identify cleavage site recognition determinants in the tRNAHis precursor. In addition, these two model substrates behave differently when comparing cleavage by M1G294 RNA. The residues immediately upstream of the –1 cleavage site in pATHis“–2U” and pATSerGC“–2A” are different, U and A, respectively (Fig. 1). To investigate the contribution of the residue at –2 to cleavage site recognition (if any), we changed the A at –2 in pATSerGC“–2A” to a U (pATSerGC“–2U”) and studied the cleavage pattern of this variant for wild type M1 RNA and M1G294 RNA. The data are shown in Fig. 3 and summarized in Table 1. The cleavage patterns of pATSerGC“–2U” and pATHis“–2U” (wild type) were very similar and significantly different when compared to cleavage of pATSerGC“–2A” by these two M1 RNA variants. This suggests that residue –2 in pATSerGC plays a significant role for cleavage site recognition. This finding suggests that changing the corresponding residue in the pATHis“–2U” (wild type) substrate from a U to an A would influence cleavage site recognition such that cleavage of the pATHis“–2A” variant would be more similar to the cleav-
age pattern of pATSerGC“–2A”. As shown in Fig. 3 and Table 1, this is what we observed. However, comparing cleavage of pATHis“–2A” and pATSerGC“–2A” by the mutant M1G294 RNA still showed a clear difference with respect to preference of cleavage at –1 vs. the +1 position (Table 1). This finding suggests that pATHis“–2U” (wild type) harbors additional determinants involved in cleavage site recognition. Based on previous findings, this might involve the residue at the –3 position i.e. the nucleotide two bases upstream of the cleavage site (see [9]). We conclude that residue –2 in the model substrates used here function as a positive determinant (but not the sole determinant) for cleavage site recognition. 3.2. Addition of Sr2+ reduces cleavage at the –1 position We previously showed that cleavage in the presence of various combinations of divalent metal ions affects cleavage site recognition such that addition of transition metal ions increases miscleavage while cleavage in the presence of Sr2+ for example results in increased frequency of cleavage at the +1 position [15,21]. Here we wanted to investigate whether the cleavage pattern of the pATSerGC and pATHis derivatives changed upon addition of Sr2+ while keeping the total divalent metal ion concentration constant at 40 mM (see Section 2). The results are shown in Fig. 3 and are summarized in Table 1.
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Fig. 2. The secondary structure of E. coli RNase P RNA, M1 RNA, according to Haas and Brown [69]. The M1C294 RNA and M1G294 RNA derivatives carry changes at position 294 as indicated while the box indicated with dashed lines represents the “P15-loop”. The boxed “GGU-motif” is part of the RCCA-RNase P RNA interaction. For details see [15,21].
In the initial experiments, we observed that cleavage of pATSerGC“–2A” at –1 by wild type M1 RNA was reduced while cleavage at the +1 position appears to be unaffected (Fig. 3 and data not shown). This was also observed when U294 in M1 RNA was substituted with a C (Fig. 3 and Table 1; see also Fig. 2). Based on the results obtained for cleavage of the other model substrate derivatives, pATSerGC“–2U”, pATHis“–2U” and pATHis“–2A”, it appears that the Sr2+ induced decrease of cleavage at the –1 position depended on the identity of residue –2. By contrast, cleavage of pATSerCG at –1 by M1G294 RNA was not decreased upon addition of Sr2+ (data not shown; note that this substrate carries a G at +73 and a G at 294 in M1 RNA).
We also investigated the significance of the 2’ OH at position +73 in the substrate because previous data indicated participation of this 2’ OH in Mg2+ binding [23,24]. Substitution to deoxy at +73, pATSerGdC“–2A”, resulted in decreased cleavage at –1 upon addition of Sr2+ but to a lesser extent (Table 1). Additionally, the mutant M1G294 RNA cleaved pATSerGdC“–2A” at a higher frequency at –1 when compared to cleavage of pATSerGC“–2A” even in the presence of Mg2+ as the only divalent metal ion. Taken together, these data suggest that addition of Sr2+ influences cleavage site recognition and that the 2’ OH at position +73 plays a role in cleavage site recognition. In addition, it appears that the identities of residues +73 and –2 in the
M. Brännvall et al. / Biochimie 84 (2002) 693–703
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Fig. 3. 5’-cleavage fragments generated after cleavage of various [γ-32P]-ATP 5’ end-labeled model substrates by different M1 RNA derivatives. The experiments were performed as described in Section 2 at 40 mM final concentration of divalent metal ions. In the experiments where Mg2+ and Sr2+ were mixed, the final concentration of each was 20 mM. The time of incubation of M1 RNA in the presence of the various model substrates is indicated. (A) Cleavage of pATHis derivatives and (B) cleavage of pATSer derivatives. The controls consisted in incubating model substrates in the presence 20 mM Mg2+ and 20 mM Sr2+ and no M1 RNA: (I) pATHis“–2U” (wild type); (II) pATHis“–2A”; (III) pATSerGdC“–2A”; (IV) pATSerGC“–2A” and (V) pATSerGC“–2U”.
substrate and 294 in M1 RNA affect the Sr2+-induced decrease of cleavage at –1. 3.3. Residue –2 influences the kinetics of cleavage at –1 Next we determined the kinetic constants for cleavage of the various substrates described above except pATSerGdC“–2A”. In our initial experiments at [Mg2+] = 40 mM where we studied cleavage by wild type M1 RNA, we were unable to reach substrate saturation when we plotted initial rates as a function of substrate
concentration. (Note the absence of the +73/294 interaction in the RS-complex for the different M1 RNA substrate combinations used in the present study, see Fig. 4.) We therefore decided to increase the Mg2+-concentration to 160 mM since it has previously been shown that an increase in Mg2+ concentration influences the kinetic constants in cleavage of model substrates [24]. At 160 mM, Mg2+ substrate saturation was observed for the various M1 RNA substrate combinations studied indicating that an increase in Mg2+ can stabilize the interaction between M1 RNA and the model substrates used in this report (see also below). As a
Table 1 Summary of frequencies of cleavage of the various substrates used in the present study. The frequencies of cleavage at –1 and at +1 are given in percentage (± experimental errors only given for cleavage at –1) and are averages of several independent experiments. The experiments were performed in the presence of 40 mM Mg2+ as described in Section 2. Note that for the tRNAHis precursor the –1 position is the natural RNase P cleavage site. Substrate
pATSerCG“–2A” pATSerGC“–2A”
pATSerGdC“–2A” pATSerGC“–2U” pATHisGC“–2U” pATHisGC“–2A” . a
M1 RNA variant identity of residue at position 294 U294 (wild type) G294 a U294 C294 G294 U294 G294 U294 G294 U294 G294 U294 G294
Numbers taken from [15].
a
Cleavage site [Mg2+] = 40 mM
Cleavage site [Mg2+] = 20 mM; [Sr2+] = 20 mM
–1
+1
–1
+1
2.4 84 90 ± 2.7 74 ± 8.4 14 ± 4.5 97 ± 0.5 40 ± 1 >99 94 ± 0.5 99 ± 0.2 99 ± 0.3 94 ± 1 75 ± 2
97.6 16 10 26 86 3 60 <1 6 1 1 6 25
55 ± 3.9 40 ± 7.2 9.5 ± 3.4 86 ± 4 21 ± 2 95 ± 2 82 ± 1 >99 98 ± 0.5 78 ± 5 56 ± 2
45 60 90.5 14 79 5 18 <1 2 22 44
698
M. Brännvall et al. / Biochimie 84 (2002) 693–703
Fig. 4. Model of RNase P RNA mediated cleavage of RNA. Top, simplified scheme of the cleavage reaction, see also [15]. Bottom, representation of RS-complexes that result in cleavage preferentially at +1 (RS’’) and at –1 (RS’), respectively. The model suggests that cleavage at +1 is preceded by two intermediates, RS’ and RS’’, and cleavage at +1 is dictated by the rate constants kchem–1 and k+2 (k–2), for details see text. In this model, a pyrimidine at –2 (or –1 when cleavage occurs at +1; boxed residues, RS’’ in A and RS’ in B, respectively) influences the rate of cleavage at the respective position by mediating a positive interaction with M1 RNA (for details see text). Three divalent metal ions are depicted in the figure, 1–3; two (2) and (3) have been localized in the P15-loop [16,35] and one (1) is positioned in the vicinity of the cleavage site [21,24,30,35]. Small arrows indicate the cleavage sites. Note that cleavage at the –1 position also occurs in the presence of the +73/294 interaction and that frequency of cleavage at –1 increases with increasing concentration of Mg2+ (Table 2) or as the result of addition of transition metals [15,21].
reference substrate, we used pATSerCG that is the “wild type” pATSer derivative [15,21]. The data are summarized in Table 2. As a result of increasing the concentration of Mg2+ we observed a threefold stimulation of the efficiency of cleavage of the reference substrate, pATSerCG, which mainly was due to a decrease in Km indicating an improvement in the interaction between the substrate and M1 RNA. In addition, an increased miscleavage at the –1 position was observed. The pATSerGC“–2A” substrate that is cleaved mainly at –1 was cleaved with a significant increase in Km (compared to cleavage of pATSerCG at –1) which corresponds to a loss of ≈1.7 kcal/mol. This was expected and is in keeping with an impaired interaction between C+73 in the
substrate and U294 in M1 RNA (see Fig. 4 and below). The wild type pATHis“–2U” substrate was cleaved approximately threefold more efficiently when compared to pATSerGC“–2A” with significant differences in both kcat and Km. Thus, the kinetics of cleavage differed when comparing cleavage of these two substrates. Substitution of residue –2 in pATSerGC“–2A” to a U (pATSerGC“–2U”) resulted in an almost 200-fold increase in kcat, while the U to A replacement in pATHis“–2A” resulted in 10- and threefold changes in kcat and Km, respectively. This discrepancy, kcat effect for pATSer vs. kcat and Km effect for pATHis, might be due to the presence of additional determinants for cleavage site recognition for cleavage of pATHis (see above) that also affect the kinetics of cleavage.
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699
Table 2 Summary of the kinetic constants, kcat and Km, in cleavage of the various model substrates by wild type M1 RNA. ∆∆G (kcal × mol–1)
Substrate
Residue “–2” in the substrate
M1 RNA pos 294
Cleavage site
kcat (min–1)
Km (µM)
kcat/Km (µM–1 × min–1)
pATSerCG a [Mg2+] = 40 mM
A
U
–1 +1
0.13 ± 0.043 4.2 ± 2.4
4.3 ± 2.4 4.3 ± 2.4
0.027 0.97
pATSerCG [Mg2+] = 160 mM pATSerGC“–2A” [Mg2+] = 160 mM pATSerGC“–2U” [Mg2+] = 160 mM pATHisGC“–2U” [Mg2+] = 160 mM pATHisGC“–2A” [Mg2+] = 160 mM .
A
U
A
U
U
U
–1 +1 –1 +1 –1
0.08 ± 0.05 1.2 ± 0.78 0.07 ± 0.04 0.007 b 13 ± 2.3
0.4 ± 0.15 0.4 ± 0.15 3.6 ± 1.6 3.6 b 4.1 ± 0.6
0.2 3.1 0.019 0.0019 b 3.2
–3.2 3
U
U
–1
4.7 ± 0.7
72 ± 7
0.065
–2.3 4
A
U
–1
0.4 ± 0.2
266 ± 120
0.0015
14
13
(1) 1 (1) 2 (+1.4) 1 (+4.6) 2
Each value is an average of several independent experiments and kcat and Km values were determined as outlined in Section 2. Superscripts (1 to 4) in the ∆∆G column refer to the ∆∆G values that were compared. a Numbers taken from Brännvall and Kirsebom, 2001 [21]. ∆∆G values were calculated according to Wells [22] and ∆∆G values given in parenthesis were calculated comparing cleavage of pATSerCG (wild type) and pATSerGC(“–2A”). b Calculation of kcat/Km for cleavage at +1 was performed based on the fact that the cleavage frequency at +1 = 10% (Table 1) and our previous findings showing that the Km is approximately the same irrespective of cleavage site [15,21].
Nevertheless, calculation of ∆∆G values reveals a significant loss of energy as a result of replacement of the –2U with an adenosine in both substrates (Table 2). Taken together, these findings demonstrate that residue –2 plays a significant role with respect both to cleavage site recognition and kinetics.
4. Discussion The data reported here extend our understanding of the cleavage site recognition process in general and the unusual cleavage of the tRNAHis precursor in particular. The unusual cleavage of the tRNAHis precursor at the –1 position has previously been shown to be due to the primary structure of the aa-stem with the G–1C+73 base pair of particular importance [7,20,25–27]. Based on the available tRNA gene sequences, it was noted that E. coli tRNA genes/transcripts preferentially carry a uridine at the position immediately upstream of the canonical RNase P cleavage site i.e. at the –1 position (–2 for tRNAHis) [8,28]. (In fact comparison of bacterial tRNA gene sequences that are available today reveals that in most bacteria the preferred nucleotide at the –1 position is a uridine (unpublished).) As shown in Table 3, where we compared the primary structures of tRNAHis genes from various bacteria and three Archaea, all, with one exception, harbor a U at –2 i.e. position immediately upstream of the RNase P cleavage site. This is rationalized by our present findings that clearly demonstrates that both the kinetics of cleavage and cleavage site recognition of the two model substrates used in the present report are influenced by the identity of residue –2 where a uridine is the preferred nucleotide among the ones tested.
4.1. Possible role(s) of residue –2 Replacement of the C–1G+73 base pair to a G–1C+73 in the model substrate pATSerCG resulted in substantial cleavage at –1. This miscleavage was reversed by changing U294 in M1 RNA to a G ([15]; this report). Substitution of C+73 in the tRNAHis precursor or cleavage of the wild type tRNAHis precursor by M1G294 RNA resulted in cleavage both at –1 and at +1 [7,25,26]. Mutational analysis of the +73/294 interaction showed that the kinetics of cleavage changed such that the rate of product release is increased in the absence of the interaction [7,20]. These data clearly suggest that residue +73 in the substrate interacts with 294 in M1 RNA and that this interaction is important for cleavage site recognition and the kinetics of cleavage. In cleavage of the model pATSer substrate, the absence of this interaction resulted in a loss of 1.4 and 4.6 kcal/mol for cleavage at –1 and at +1, respectively, corresponding roughly to a base pair. Additionally, our recent data suggest that the interaction between +73 and 294 is stabilized by Mg2+ [21]. Moreover, in almost half of the tRNA precursors in E. coli, the residue at –1 is paired with the base at position +73 i.e. the discriminator position. Therefore, it is most likely that the residue at –1 interacts with M1 RNA as a result of formation of the +73/294 interaction in the RS-complex as discussed elsewhere [9]. This would expose the cleavage site as we have suggested previously [5]. We recently suggested that cleavage by M1 RNA proceeds via intermediates according to the simplified scheme depicted in Fig. 4 [15] . In this model, cleavage at –1 is dictated by the rate constants kchem–1 and k+2 (and k–2). The former represents the constant for the chemistry of cleavage while the latter is the rate constant for breaking the –1/+73
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Table 3 Distribution of bases at the RNase P cleavage site on the tRNAHis precursor. Organism Bacillus subtilis 168 Bacillus subtilis 168 Borrelia burgdorferi B31 Campylobacter jejuni NCTC 11168 Clostridium perfringens 13 Escherichia coli K12-MG1655 Haemofilus influenczae Rd (KW20) Listeria monocytogenes EGD-e Mycobacterium leprae TN Mycobacterium tuberculosis H37Rv Mycoplasma genitalium G-37 Mycoplasma pneumoniae M129 Neisseria meningitideis MC58 Pseudomonas aeruginosa PA01 Rickettsia prowazekii Madrid E Salmonella typhimurium LT2 Staphylococcus aureus Mu50 Streptomyces coelicolor A3(2) Thermatoga maritima MSB8 Treponema palladiium Nichols Vibrio cholerae El Tor N16961 Vibrio cholerae El Tor N16961 Yersinia pestis CO92 Archaeoglobus fulgidus DSM4304 Methanococcus jannaschii DSM2661 Sulfolobus solfataricus P2 . a b
tRNA a
trnB trnD a
b
b
b
His1 His2
Position –2
Position –1
Reference
U U U U U U U U U U U U U U U U U U U C U U U U U U
G G G G G G G G G G G G G G G G G G G G G G G G G G
[46] [46] [45] [67] [68] [47] [55] [63] [49] [51] [48] [50] [56] [52] [53] [58] [57] [62] [59] [60] [54] [54] [61] [64] [65] [66]
Both tRNAs are identical except for a C to U change in the variable loop. Harbors two genes annotated as tRNAHis genes.
base pair in the substrate and forming the +73/294 interaction in the RS-complex. Thus, a slow k+2 (due to the absence of the +73/294 interaction) will favor cleavage at the –1 position as in cleavage of pATSerGC“–2A” by wild type M1 RNA. By contrast, when k+2 is fast i.e. the +73/294 interaction is established (going from RS’ to RS’’; Fig. 4) as in the case of cleavage of pATSerGC“–2A” with M1G294 RNA, then cleavage will preferentially occur at the +1 position. Replacement of the –2A with a –2U in pATSerGC“–2A” resulted in a significant increase in kcat (Table 2). Product release is apparently not the rate-limiting step in cleavage of this type of model substrates [24]. Hence, the given kcat values most likely describe the reaction prior to release of the product(s). The increase in kcat also indicates a role for residue –2 going from the ground state to the transition state. This is associated with a change in ∆∆G of 3.2 kcal/mol (–2A compared to –2U; Table 2), which roughly corresponds to formation/disruption of a base pair interaction. Thus, a likely possibility is that a U at the –2 position affects the rate of cleavage by mediating a positive interaction with M1 RNA such that kcat is increased (Fig. 4). In this context, we have previously discussed the possibility that a U at –1 (which corresponds to –2 in tRNAHis) might interact with the J18/2 region (A328–U335) ([8]; see also [29]). However, the J5/15 (A248, A249) and P15 (C252, C253) regions must also be considered since cross-linking data suggest that residues in these regions are in close proximity to residue –1 [8]. Alternatively, but not mutually
exclusive, a U immediately upstream of the cleavage site might affect coordination of (a) Mg2+-ion(s) important for the chemistry of cleavage. In fact it is clear that Mg2+ binds in the vicinity of the RNase P cleavage site [21,23,24,30]. In this context, we note that the “wild type” pATSerCG substrate was miscleaved at a higher frequency at elevated Mg2+ concentrations (Table 1 and data not shown). At present we cannot distinguish between these possibilities or exclude other explanations. For example we note that for in vitro modified yeast tRNAPhe precursors the 2’ OH immediately upstream of the canonical RNase P cleavage site was suggested to mediate an interaction with RNase P RNA that plays a role for positioning of the scissile bond in the active site [12]. Together with our findings, this scenario would suggest that the identity of residue –2 influences formation of the (–2) “2’ OH–M1 RNA” interaction and/or positioning of the scissile bond. Clearly, more detailed experiments have to be performed to establish the function of the nucleotide immediately upstream of the cleavage site in the RNase P RNA catalyzed reaction. 4.2. Importance of the 2’ OH at position +73 in the substrate Several 2’ OH in the substrate have been implicated to play a role in the interaction with RNase P RNA and in coordination with Mg2+ [8,23,24,31–35]. Among these 2’ OH groups, the 2’ OH at the +73 position has been suggested to be involved in coordination of Mg2+ [23,36].
M. Brännvall et al. / Biochimie 84 (2002) 693–703
Here we showed that removal of the 2’ OH at +73 resulted in an increased frequency of cleavage at the –1 position when compared to cleavage in its presence (Table 2; compare cleavage of pATSerGC“–2A” and pATSerGdC“–2A” with M1G294 RNA). This is in keeping with data where we studied cleavage of other pATSer derivatives by wild type M1 RNA (to be published elsewhere). Moreover, addition of Sr2+ appeared to result in a reduction of cleavage of pATSerGC“–2A” by wild type M1 RNA preferentially at –1. In the absence of the 2’ OH at +73, the Sr2+-induced decrease of cleavage at –1 was less accentuated (Table 2). A structural study of a model of tRNAAla showed that Sr2+ coordinates to 2’ OH groups [37]. It is therefore likely that Sr2+ utilize the 2’ OH at +73 as a ligand and thereby displace Mg2+. As a consequence, this results in a reduced rate of cleavage at –1. Taken together, our present findings lend support to the involvement of the 2’ OH at +73 in Mg2+ binding and suggest that this Mg2+ influence cleavage site recognition. In view of our recent findings [21], this Mg2+ is likely to be involved in metal ion cooperativity in cleavage by RNase P RNA. 4.3. Expression of tRNA genes is influenced by structural elements localized outside the promoter region Substitution of the nucleotide at the +2 position in tRNATyrSu3 from a G to an A results in a reduction of the intracellular concentration of 5’ matured tRNA. The reduction appears to be complemented by a C to U change at position –4 in the leader [38]. This finding might be rationalized in view of more recent data that suggest that the RNase P protein is positioned close to the cleavage site and enhances the interaction with the 5’ leader ([39]; see also [29,40]). Thus, it is possible that substitution of residue –4 influences this interaction and thereby RNase P cleavage efficiency. Moreover, in vitro studies with Bacillus subtilis RNase P demonstrate that the length of the 5’ leader influenced the catalytic efficiency ([41]; see also [42]). Krupp et al. [42] also noted that cleavage between pyrimidine–purine bonds was preferred relative to cleavage of purine–purine bonds. Recently it was demonstrated that a C to A (or G) change in a model substrate reduced cleavage for the RNase P holoenzyme derived from E. coli as well as B. subtilis [43] consistent with our present findings (Table 2). These in vitro studies are in keeping with our tRNATyrSu3 nonsense suppressor data where replacement of –1U to –1A resulted in a significant reduction in suppression efficiency [44] as well as the low appearance of a purine at the –1 position in tRNA genes/transcripts. Finally, cleavage of various tRNA-like structures with RNase P RNA in the presence/absence of the RNase P protein suggests that the nucleotide at the position corresponding to the –1 position in a tRNA precursor plays an important role in the reaction [28]. In conclusion, we suggest that the nucleotide immediately upstream of the cleavage site is a positive determinant
701
for cleavage by RNase P. In addition, expression of tRNA genes is influenced by structural elements localized outside the promoter region i.e. in the leader and spacer regions of tRNA transcripts. Based on this and the data reported here raise the possibility that a U to A change at –2 in the tRNAHis gene in for example E. coli might lower expression of tRNAHis.
Acknowledgements We thank our colleagues for discussions, Dr. S. Dasgupta for critical reading of the manuscript. This work was supported by grants from the Swedish Natural Science Research Council, the Foundation for Strategic Research and the Wallenberg Foundation (to L.A.K.).
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