Cleavage of Model Substrates by Arabidopsis thaliana PRORP1 Reveals New Insights into Its Substrate Requirements
2016 (English)In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 11, no 8, e0160246Article in journal (Refereed) Published
Two broad classes of RNase P trim the 5' leader of precursor tRNAs (pre-tRNAs): ribonucleoprotein (RNP)- and proteinaceous (PRORP)-variants. These two RNase P types, which use different scaffolds for catalysis, reflect independent evolutionary paths. While the catalytic RNA-based RNP form is present in all three domains of life, the PRORP family is restricted to eukaryotes. To obtain insights on substrate recognition by PRORPs, we examined the 5' processing ability of recombinant Arabidopsis thaliana PRORP1 (AtPRORP1) using a panel of pre-tRNA(Ser) variants and model hairpin-loop derivatives (pATSer type) that consist of the acceptor-T-stem stack and the T-/D-loop. Our data indicate the importance of the identity of N-1 (the residue immediately 5' to the cleavage site) and the N-1: N+73 base pair for cleavage rate and site selection of pre-tRNA(Ser) and pATSer. The nucleobase preferences that we observed mirror the frequency of occurrence in the complete suite of organellar pre-tRNAs in eight algae/plants that we analyzed. The importance of the T-/D-loop in pre-tRNA(Ser) for tight binding to AtPRORP1 is indicated by the 200-fold weaker binding of pATSer compared to pre-tRNA(Ser), while the essentiality of the T-loop for cleavage is reflected by the near-complete loss of activity when a GAAA-tetraloop replaced the T-loop in pATSer. Substituting the 2'-OH at N-1 with 2'-H also resulted in no detectable cleavage, hinting at the possible role of this 2'-OH in coordinating Mg2+ ions critical for catalysis. Collectively, our results indicate similarities but also key differences in substrate recognition by the bacterial RNase P RNP and AtPRORP1: while both forms exploit the acceptor-T-stem stack and the elbow region in the pre-tRNA, the RNP form appears to require more recognition determinants for cleavage-site selection.
Place, publisher, year, edition, pages
2016. Vol. 11, no 8, e0160246
IdentifiersURN: urn:nbn:se:uu:diva-307895DOI: 10.1371/journal.pone.0160246ISI: 000381369500026OAI: oai:DiVA.org:uu-307895DiVA: diva2:1048798
FunderSwedish Research Council, Dnr 349-2006-267 Dnr 621-2011-5848Carl Tryggers foundation
De två första författarna delar förstaförfattarskapet.2016-11-222016-11-222016-11-22Bibliographically approved