Substrate discrimination in RNase P RNA-mediated cleavage: importance of the structural environment of the RNase P cleavage site

Abstract

Like the translational elongation factor EF-Tu, RNase P interacts with a large number of substrates where RNase P with its RNA subunit generates tRNAs with matured 5′ termini by cleaving tRNA precursors immediately 5′ of the residue at +1, i.e. at the position that corresponds to the first residue in tRNA. Most tRNAs carry a G+1C+72 base pair at the end of the aminoacyl acceptor-stem whereas in tRNAGln G+1C+72 is replaced with U+1A+72. Here, we investigated RNase P RNA-mediated cleavage as a function of having G+1C+72 versus U+1A+72 in various substrate backgrounds, two full-size tRNA precursors (pre-tRNAGln and pre-tRNATyrSu3) and a model RNA hairpin substrate (pATSer). Our data showed that replacement of G+1C+72 with U+1A+72 influenced ground state binding, cleavage efficiency under multiple and single turnover conditions in a substrate-dependent manner. Interestingly, we observed differences both in ground state binding and rate of cleavage comparing two full-size tRNA precursors, pre-tRNAGln and pre-tRNATyrSu3. These findings provide evidence for substrate discrimination in RNase P RNA-mediated cleavage both at the level of binding, as previously observed for EF-Tu, as well as at the catalytic step. In our experiments where we used model substrate derivatives further indicated the importance of the +1/+72 base pair in substrate discrimination by RNase P RNA. Finally, we provide evidence that the structural architecture influences Mg2+ binding, most likely in its vicinity.

Eukaryotic RNase P RNA mediates cleavage in the absence of protein

“…pATSerUG was either purchased from Dharmacon USA (Lafayette, CO) or prepared as run-off transcripts by using T7 DNA-dependent RNA polymerase. This model substrate is cleaved as efficiently as precursor tRNAs (12). The different tRNA precursors were generated as run-off transcripts (26)(27)(28).…”

Characterization of a cyanobacterial RNase P ribozyme recognition motif in the IRES of foot-and-mouth disease virus reveals a unique structural element

“…8). This situation may be reminiscent of the modified enzyme-substrate interaction described in the 39-CCA region of the tRNA precursor (Kikovska et al 2005).…”

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

“…22 The structural topography of the + 73/294 interaction can influence the cleavage and binding of functionally important metal(II) ions (e.g., Mg 2+ ) at and in the vicinity of the cleavage site. 19,29,30,[33][34][35][36][37][38] This therefore raises the possibility that a productive TSL/TBS interaction, required for efficient cleavage at the correct site, involves the positioning of metal(II) ions near the cleavage site (see the text below). In this context, we also note that increasing the size of the T-loop in SELEX (Selective Evolution of Ligands by EXponential enrichment)-evolved substrates lowers the cleavage efficiency (k cat /K m ) for EcoRPR.…”