Crystallization and crystallographic analysis of an Arabidopsis nuclear proteinaceous RNase P

RNase P activity is ubiquitous and involves the 5' maturation of precursor tRNAs. For a long time, it was thought that all RNases P were ribonucleoproteic enzymes. However, the characterization of RNase P in human mitochondria and in plants revealed a novel kind of RNase P composed of protein only, called PRORP for `proteinaceous RNase P'. Whereas in human mitochondria PRORP has two partners that are required for RNase P activity, PRORP proteins are active as single-subunit enzymes in plants. Three paralogues of PRORP are found in Arabidopsis thaliana. PRORP1 is responsible for RNase P in mitochondria and chloroplasts, while PRORP2 and PRORP3 are nuclear enzymes. Here, the purification and crystallization of the Arabidopsis PRORP2 protein are reported. Optimization of the initial crystallization conditions led to crystals that diffracted to 3 Å resolution

Caractérisation biochimique et structurale des RNases P et MRP chez la levure Saccharomyces cerevisiae

La RNase P est une endoribonucléase responsable de la maturation de l extrémité 5 des ARNt prématures. Holoenzyme très conservée, elle est constituée d une composante ARN formant le noyau catalytique et d une composante protéique dont le nombre de sous-unités est variable : une protéine chez les bactéries, 5 chez les archées et d au moins 9 chez les eucaryotes. Les eucaryotes possèdent également une autre endoribonucléase, la RNase MRP dont la composition est proche de la RNase P tant au niveau ribonucléique que protéique mais avec une spécificité de substrat propre. Dans cette étude, nous proposons une méthode originale et spécifique pour purifier la RNase P et la RNase MRP de S. cerevisiae. Grâce à la microscopie électronique et au traitement d images, nous avons déterminé la première structure de ces deux holoenzymes à une résolution d environ 1.5 nm. Ces structures révèlent une architecture modulaire commune où les protéines stabilisent la composante ARN et contribuent à l édification de cavités et de conduits. Les spécificités structurales sont localisées en des positions stratégiques pour l identification et la coordination du substrat.Ribonuclease P (RNase P) is an endoribonuclease that cleaves the 5'-leader sequence of pre-tRNAs. RNase P is conserved between all taxonomic kingdoms and consists of a catalytic RNA subunit and protein components of variable size, from one protein in bacteria to 5 proteins in archae and at least 9 proteins in eukaryotic cells. In addition to RNase P, eukaryotes possess the RNase MRP which has a related RNA core and shares 8 proteins subunits with RNase P but with its own substrate specificity. Here, we propose an original method to purify specifically RNase P and RNase MRP from S. cerevisiae. Using electron microscopy and image processing, we solved the first structure of these two holoenzymes at a resolution of about 1.5 nm. We showed that eukaryotic RNase P and RNase MRP have a modular architecture, where proteins stabilize the RNA fold and contribute to cavities, channels and chambers between the modules.Structural features are located at strategic positions for substrate recognition by shape and coordination of the cleaved-off sequence.STRASBOURG-Bib.electronique 063 (674829902) / SudocSudocFranceF

Distribution of ribonucleoprotein and protein-only RNase P in Eukarya

RNase P is the endonuclease that removes 5' leader sequences from tRNA precursors. In Eukarya, separate RNase P activities exist in the nucleus and mitochondria/plastids. Although all RNase P enzymes catalyse the same reaction, the different architectures found in Eukarya range from ribonucleoprotein (RNP) enzymes with a catalytic RNA and up to 10 protein subunits to single-subunit protein-only RNase P (PRORP) enzymes. Here, analysis of the phylogenetic distribution of RNP and protein-only RNase P enzymes in Eukarya revealed (i) a wealth of novel P RNAs in previously unexplored phylogenetic branches and (ii) that PRORP enzymes are more widespread than previously appreciated, found in four of five eukaryal supergroups, in the nuclei and/or organelles. Intriguingly, the occurrence of RNP RNase P and PRORP seems mutually exclusive in genetic compartments of modern Eukarya. Our comparative analysis provides a global picture of the evolution and diversification of RNase P throughout Eukarya

Biophysical analysis of Arabidopsis protein-only RNase P alone and in complex with tRNA provides a refined model of tRNA binding

RNase P is a universal enzyme that removes 5' leader sequences from tRNA precursors. The enzyme is therefore essential for maturation of functional tRNAs and mRNA translation. RNase P represents a unique example of an enzyme that can occur either as ribonucleoprotein or as protein alone. The latter form of the enzyme called PRORP (PRotein-Only RNase P) is widespread in eukaryotes, in which it can provide organellar or nuclear RNase P activities. Here, we have focused on Arabidopsis nuclear PRORP2 and its interaction with tRNA substrates. Affinity measurements helped assess the respective importance of individual pentatricopeptide repeat motifs in PRORP2 for RNA binding. We characterized the PRORP2 structure by X-ray crystallography and by small-angle X-ray scattering (SAXS) in solution, as well as that of its complex with a tRNA precursor by SAXS. Of note, our study reports the first structural data of a PRORP-tRNA complex. Combined with complementary biochemical and biophysical analyses, our structural data suggest that PRORP2 undergoes conformational changes to accommodate its substrate. In particular, the catalytic domain and the RNA binding domain can move around a central hinge. Altogether, this work provides a refined model of the PRORP-tRNA complex that illustrates how protein-only RNase P enzymes specifically bind tRNA and highlights the contribution of protein dynamics to achieve this specific interaction