Metal ion cooperativity in ribozyme cleavage of RNA

Abstract

Combinations of chemical and genetic approaches were used to study the function of divalent metal ions in cleavage of RNA by the ribozyme RNase P RNA. We show that different divalent metal ions have differential effects on cleavage site recognition and rescue of cleavage activity by mixing divalent metal ions that do not promote cleavage by themselves. We conclude that efficient and correct cleavage is the result of cooperativity between divalent metal ions bound at different sites in the RNase P RNA-substrate complex. Complementation of a mutant RNase P RNA phenotype as a result of divalent metal ion replacement is demonstrated also. This finding together with other data indicate that one of the metal ions involved in this cooperativity is positioned near the cleavage site. The possibility that the Mg 2؉ ͞Ca 2؉ ratio might regulate the activity of biocatalysts that depend on RNA for activity is discussed.RNase P ͉ divalent metal ions ͉ tRNA precursors ͉ tRNA processing D ivalent metal ions are important for the function of many proteins and RNA. The role of the divalent metal ions can be structural and͞or catalytic, and in RNA-catalyzed reactions the existence of several categories of divalent metal ion binding sites has been suggested (1-5). The reaction catalyzed by the ribozyme RNase P RNA, the catalytic subunit of the endoribonuclease RNase P (6), requires divalent metal ions; among the ones studied thus far, Mg 2ϩ promotes cleavage most efficiently. The roles of Mg 2ϩ in the RNase P RNA-catalyzed reaction are to induce proper folding of the RNA, to facilitate the interaction with its RNA substrate, and to promote efficient and correct cleavage (7). Cleavage by RNase P RNA has been reported to proceed in the presence of Mn 2ϩ and Ca 2ϩ , although at reduced efficiencies (2,(8)(9)(10)(11)(12)(13)(14)(15). However, cleavage in the presence of Mn 2ϩ and Ca 2ϩ induces miscleavage (11,12,15). These findings make RNase P RNA a suitable model system to study the way by which different divalent metal ions influence RNA-mediated cleavage and thereby expand our knowledge about the role of different categories of divalent metal ion binding sites in the RNase P RNA-catalyzed reaction and in RNA in general.Here we present data demonstrating RNase P RNA cleavage in the presence of divalent metal ions that do not promote cleavage by themselves, indicating metal ion cooperativity. Our data further suggest that one of the metal ions involved in this cooperativity is positioned in the vicinity of the interaction between the 3Ј end of the substrate and RNase P RNA, the ''RCCA-RNase P RNA'' interaction (interacting residues underlined, ref. 16). Materials and MethodsThe substrates, pATSerCG and pATSerUA, were purchased from Xeragon AG, Switzerland, or generated by run-off transcription using T7 DNA-dependent RNA polymerase (17). Internally labeled pATSerCG, 5Ј end-labeled pATSerUA, and the M1 RNA variants were generated as described elsewhere (15,18).Cleavage reactions were performed at 37°C as described in detail elsewhere (1...

NMR and XAS reveal an inner-sphere metal binding site in the P4 helix of the metallo-ribozyme ribonuclease P

“…This is expected because CoðNH 3 Þ 3þ 6 has a higher charge than Mg 2þ (28); similarly, lower concentrations of CoðNH 3 Þ 3þ 6 compared to Mg 2þ are required to stabilize the PRNA structure (7,21). Whereas Mg 2þ perturbations include specific residues above and below the U7 bulge, CoðNH 3 Þ 3þ 6 induces shifts in the bulged U7 as well as in a larger number of residues above and below this nucleotide (Fig.…”

“…CoðNH 3 Þ 3þ 6 and Zn 2þ were employed to mimic outer-and inner-sphere interactions of Mg 2þ , respectively (27). Although RNase P has no catalytic activity in the presence of CoðNH 3 Þ 3þ 6 alone (despite formation of a RNase P•pre-tRNA complex), the activity increases upon addition of either Mg 2þ or Zn 2þ (21). Under these conditions, we used NMR chemical shift perturbations to analyze the location of the inner-sphere Zn 2þ metal observed by XAS.…”

“…We also use a metal cocktail NMR chemical shift perturbation approach for resolving inner-versus outer-sphere interactions, and to corroborate the XAS results. Our approach takes advantage of the fact that RNase P is active in vitro in the presence of a variety of divalent metal ions (Mg 2þ , Ca 2þ , Mn 2þ , and Zn 2þ ∕CoðNH 3 Þ 3þ 6 ) (20,21). XAS data indicate that Zn 2þ is involved in inner-sphere interactions with the P4 helix mimic and that the bound Zn 2þ exhibits six-coordinate geometry with an average Zn 2þ -O∕N bond distance of ∼2.08 Å.…”

Functional Coupling between a Distal Interaction and the Cleavage Site in Bacterial RNase-P-RNA-Mediated Cleavage

“…Replacing U at position 294 with C seems to facilitate the disruption of the C − 1 /G + 73 pair. 19,30 We argued that the RPR C294 variant would therefore cleave the tetraloop substrates with a higher frequency at + 1 relative to the wt. To test this, we cleaved the different substrates with RPR C294 .…”

Insights into functional modulation of catalytic RNA activity

“…In another study, several divalent metal ions (Mg 21 , Mn 21 , Ca 21 and Sr 21 ) have differential effects on cleavage site recognition and rescue of bacterial RNase P RNA enzymatic activity. It seems that efficient and correct cleavage is the result of co-operativity between divalent metal ions bound at different sites in the RNase P RNA-substrate complex (58). Mg 21 and Ca 21 are relatively abundant inside the cell and regulate a wealth of physiological functions.…”