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. 2004 Oct 11;32(18):5418-29.
doi: 10.1093/nar/gkh883. Print 2004.

Cross talk between the +73/294 interaction and the cleavage site in RNase P RNA mediated cleavage

Mathias Brännvall  1 , Ema Kikovska, Leif A Kirsebom
Affiliations

Cross talk between the +73/294 interaction and the cleavage site in RNase P RNA mediated cleavage

Mathias Brännvall et al. Nucleic Acids Res. .

Abstract

To monitor functionally important metal ions and possible cross talk in RNase P RNA mediated cleavage we studied cleavage of substrates, where the 2'OH at the RNase P cleavage site (at -1) and/or at position +73 had been replaced with a 2' amino group (or 2'H). Our data showed that the presence of 2' modifications at these positions affected cleavage site recognition, ground state binding of substrate and/or rate of cleavage. Cleavage of 2' amino substituted substrates at different pH showed that substitution of Mg2+ by Mn2+ (or Ca2+), identity of residues at and near the cleavage site, and addition of C5 protein influenced the frequency of miscleavage at -1 (cleavage at the correct site is referred to as +1). From this we infer that these findings point at effects mediated by protonation/deprotonation of the 2' amino group, i.e. an altered charge distribution, at the site of cleavage. Moreover, our data suggested that the structural architecture of the interaction between the 3' end of the substrate and RNase P RNA influence the charge distribution at the cleavage site as well as the rate of cleavage under conditions where the chemistry is suggested to be rate limiting. Thus, these data provide evidence for cross talk between the +73/294 interaction and the cleavage site in RNase P RNA mediated cleavage. We discuss the role metal ions might play in this cross talk and the likelihood that at least one functionally important metal ion is positioned in the vicinity of, and use the 2'OH at the cleavage site as an inner or outer sphere ligand.

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Figures

Figure 1
Figure 1
The predicted secondary structure of the model substrate pATSerUG is depicted. The substitutions that resulted in the pATSer variants are indicated and the stars (*) indicate where the 2′N (or 2′H) modifications were introduced. The residue immediately preceding the 3′ CCA motif is referred to as residue +73 since it corresponds to residue +73 in precursor tRNA. The arrows indicate cleavage sites and the inset shows where the 2′N (or 2′H) was inserted.
Figure 2
Figure 2
Cleavage site recognition of different pATSer model substrates under different conditions as indicated. [γ-32P]ATP 5′ end-labeled substrates were cleaved and only the 5′ cleavage fragments are shown. The cleavage sites are indicated as +1 and −1. (A) Cleavage of pATSerUA and pATSerUamA in the presence of different divalent metal ions as indicated under our standard assay conditions at pH 7.2 (for pATSerUG, cleavage was performed at pH 5.5, 6.5, 7.2 and 8.6). The concentrations of M1 RNA and substrates were 0.24 and ≤0.05 μM, respectively. As controls, substrate and M1 RNA were incubated for 180 min in the presence of Co(NH3)63+ and Sr2+ [neither of these alone can promote cleavage (6)]. Time of incubation for cleavage of pATSerUA was 8 min in the presence of Mg2+ and Mn2+ and 180 min in the presence of Ca2+; pATSerUamA was 180 min irrespective of metal ion and; pATSerUG was 40 min (note in the presence of 40 mM Mg2+). (B) Cleavage of pATSerUamG, pATSerUamA, pATSerUamU and pATSerCamG in the presence of different divalent metal ions and different pH as indicated (increasing pH from left to right). The experiments were performed in 50 mM Bis-Tris Propane buffer at pH 5.5, 6.1, 6.5, 6.9, 7.2, 8.1 and 8.6 in the presence of 40 mM divalent metal ion Mg2+, Mn2+ or Ca2+. Cleavage of pATSerUamA in the presence of Mn2+ or Ca2+ was performed at pH 5.6, 6.4, 6.8, 7.1 and 7.4, respectively. The concentrations of M1 RNA and substrates were 0.16 and ≤0.05 μM, respectively (in the case of cleavage of pATSerUamA, 0.19 μM and 0.08 μM, respectively). Time of incubation in the presence of: Mg2+ = 260 min; Mn2+ = 285 min; Ca2+ = 262 min. For pATSerUamA, the incubation time in the presence of Mn2+ and Ca2+ was 180 min. Ctrl = control in the presence of Ca2+ with no M1 RNA added. (C) Cleavage of pATSerUG, pATSerdUG, pATSerUamA, pATSerUamG, pATSerCG and pATSerCamG in the presence of 160 mM Mg2+ at different pH as indicated (increasing pH from left to right). Same buffer conditions (50 mM Bis-Tris Propane, 5% (w/v) PEG 6000, 100 mM NH4Cl and 160 mM MgCl2) and pH as described in Figure 2B were used for cleavage of these substrates except pATSerUG, which was cleaved at pH 5.0, 6.1, 7.3 and 8.5 with the same buffer conditions. The concentrations of M1 RNA and substrates were 0.19 and 0.08 μM, respectively (in cleavage of pATSerUG, 0.04 μM substrate and 1.31 μM M1 RNA, while for cleavage of pATSerdUG, pATSerCG and pATSerCamG 0.02 μM substrate and 1.02 μM M1 RNA were used). Time of incubation for cleavage of: pATSerUG, 15 min; pATSerdUG, 150 min; pATSerUamA and pATSerUamG, 265 min; pATSerCG, 50 min; pATSerCamG, 150 min. In the experiments using pATSerUamA and pATSerUamG, the reactions were terminated by the addition of 99% ethanol. The precipitates were resolved in loading buffer as described elsewhere (23). (D) Cleavage of pATSerUamA and pATSerUamG in the presence of the C5 protein at different pH as indicated (increasing pH from left to right). Cleavage of pATSerUamA was performed in the presence of 50 mM MES and 50 mM Tris–HCl buffers (see Materials and Methods) at 20 mM Mg2+ while cleavage of pATSerUamG was performed in 50 mM Bis-Tris Propane buffer at pH 6.6, 7.1, 8.2 and 9.1 and 10 mM Mg2+. The concentrations of M1 RNA and substrates were 0.0024 and ≤0.05 μM, respectively, irrespective of substrate. The C5 protein was added in excess as previously described (28). Time of incubation for cleavage of pATSerUamA, and pATSerUamG was 210 and 227 min, respectively. The controls were: no C5 protein, 0.24 μM M1 RNA incubated with ≤0.05 μM pATSerUamA for 210 min; no M1 RNA, substrate incubated at 40 mM Mg2+ in the absence of M1 RNA and C5 protein for 210 and 227 min, respectively.
Figure 3
Figure 3
Frequency of cleavage at −1 of pATSerUG, pATSerdUG and pATSerUamG as a function of pH in the presence of different divalent metal ions as indicated. The curves are averages of several independent experiments and the bars indicate experimental errors. The experiments were performed at 37°C as outlined in Materials and Methods. Data shown for pATSerUG and pATSerdUG were taken from experiments performed at 160 mM Mg2+.
Figure 4
Figure 4
The predicted secondary structure of E.coli RNase P RNA (M1 RNA) according to Haas and Brown (63). The broken-line box represents the P15-loop while the boxed GGU-motif represents the residues that base pair with the 3′ RCCA motif of the substrate – the RCCA-RNase P RNA interaction – as illustrated in the inset. The M1 RNA variants C294, A294 and G294 used in this study are indicated. The inset illustrates a model of the RCCA-RNase P RNA interaction. Here A,B and C (encircled) represent divalent metal ions—for details see text and (19). The U at position 294 is highlighted in black.
Figure 5
Figure 5
Cleavage as a function of the structural architecture of the +73/294 interaction. The experiments were conducted in buffer B in the presence of 40 mM at indicated pH under single turnover conditions (see Materials and Methods). Given numbers refer to the frequency of cleavage at +1 and −1. The controls represent incubation of indicated substrates in buffer B and 40 mM Mg2+ in the absence of M1 RNA. (A) Cleavage of pATSerUamG with wild type and M1C294 RNA. (B) Cleavage of pATSerUamU and pATSerUamUam with wild type and M1G294 RNA.
Figure 6
Figure 6
Model of the cleavage site where the upper part illustrates the −1, +1, +72 and +73 in the substrate as indicated. The RCCA-RNase P RNA interaction between M1 RNA and the 3′ end of the substrate is shown in dashed lines (see Figure 4). The dashed line with double arrow heads indicate cross talk between Mg2+, positioned in the vicinity of the interaction between residues +73 in the substrate and U294 in M1 RNA (metal ion A in Figure 4), and the cleavage site including MgB2+ (metal ion B in Figure 4). As outlined in the text, at present we cannot distinguish whether the 2′OH is engaged in an inner or outer sphere interaction with MgB2+.
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References

    1. Doudna J.A. and Cech,T.R. (2002) The chemical repertoire of natural ribozymes. Nature, 418, 222–228. - PubMed
    1. Guerrier-Takada C., Gardiner,K., Marsh,T., Pace,N. and Altman,S. (1983) The RNA moiety of ribonuclease P is the catalytic subunit of the enzyme. Cell, 35, 849–857. - PubMed
    1. Altman S. and Kirsebom,L.A. (1999) Ribonuclease,P. In Gesteland,R.F., Cech,T.R. and Atkins,J.F. (eds.), The RNA World, 2nd edn. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, pp. 351–380.
    1. Vioque A., Arnez,J. and Altman,S. (1988) Protein-RNA interactions in the RNase P holoenzyme from Escherichia coli. J. Mol. Biol., 202, 835–848. - PubMed
    1. Fang X.W., Yang,X.J, Littrell,K., Niranjanakumari,S., Thiyagarajan,P., Fierke,C.A., Sosnick,T.R. and Pan,T. (2001) The Bacillus subtilis RNase P holoenzyme contains tow RNase P RNA and two RNase P protein subunits. RNA, 7, 233–241. - PMC - PubMed

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