Functional characterisation of the novel protein-only RNase P from Arabidopsis thaliana – differences to ribozyme-based bacterial RNase P
Abstract
Ribonuclease P has been predominantly identified as a ribonucleoprotein that is responsible for the 5'-maturation of precursor tRNA (ptRNA). RNA-containing RNase P enzymes are present in all domains of life (Bacteria, Archaea and Eukarya). The majority of RNase P enzymes consists of one RNA subunit and at least one or up to ten protein subunits. The RNA moiety has been shown to be the catalytic subunit of the ribonucleoprotein enzymes [1-4].
Notwithstanding this general view, studies on RNase P activities from the organelles of some Eukaryotes are at odds with a general ribonucleoprotein nature of the enzyme. Human mitochondrial and spinach chloroplast RNase P are composed of protein only [5,6]. The same findings were reported for the trypanosomal mitochondrial RNase P [7], and recently three protein-only RNase P enzymes were identified in Arabidopsis thaliana [8], of which one (PRORP1) localizes to mitochondria and chloroplasts, whereas the other two (PRORP2 and PRORP3) are exclusively present in the nucleus. Recent data have demonstrated that Arabidopsis PRORP1 is able to replace the bacterial ribonucleoprotein in vivo [8,9].
The goal of this project is to characterise the function of the novel protein-only RNase P enzymes PRORP1, PRORP2 and PRORP3 from Arabidopsis in vitro and in vivo. First, these protein enzymes have been compared with the classical RNA-based RNase P enzymes from bacteria in terms of substrate recognition, kinetic parameters and cleavage mechanism. Beside several bacterial and plant organellar ptRNA substrates, Rp-/Sp-phosphorothioate-modified ptRNAs and minimal substrates are used for the in vitro studies. Furthermore, the E. coli complementation strain, in which PRORP1 replaces the endogenous RNA-based RNase P and which shows somewhat retarded growth, was investigated by transcriptome analysis (dRNA-Seq / Solexa) in order to elucidate how PRORP1 acts on endogenous E. coli tRNA and non-tRNA substrates.
References
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