La maturation des ARNt en 5' est réalisée par RNase P. C'est un ribozyme chez les bactéries, les fungi et les nuclei des mammifères et un enzyme protéique dans les plantes ou des organelles des mammifères qui s’appelle PRORP. Il y a trois PRORP dans A. thaliana. PRORP contiennent deux domaines : un domaine PPR qui reconnaît spécifiquement des séquences d'ARN et un domaine nucléase qui assure la coupure endonucléolytique 5' des précurseurs d’ARNt. Pendant ma thèse j'ai pu montré par des méthodes biophysiques et structurales comme SRCD et SAXS que PRORP1 et 2 sont composées en majorité des hélices alpha Elles ont un rayon de giration de 33 Å et contiennent deux domaines distincts avec et une dimension maximale de 110 Å. Pour le complex entre un substrat d'ARNt et PRORP une constante de dissociation de 1 uM a pu être confirmé par la microcalorimétrie, la thermophorèse et l'ultracentrifugation analytique. Ces analyses nous ont permis de construire un modèle PRORP et un substrat d'ARNt. / RNase P cleaves 5’ leaders of precursor tRNAs. RNase P is a ribozyme in bacteria, fungi and animal nuclei and a protein in animal organelles, plants and many other organism. There are three PRORPs in A. thaliana. MALS, SRCD and SAXS provided first structural information: 1) PRORPs are monomers in solution. 2) PRORP 1-2 have a high alpha-helical content. 3) PRORPs are composed of two distinct domains with a radius of gyration of 33 A. These results together with homology modelling enabled us to build a first model of PRORPs in complex with tRNA. Using three different methods, isothermal titration calorimetry, microscale thermophoresis and analytical ultracentrifugation, a binding constant of about 1 µM could be determined for the system PRORP2mDD and L5T0 tRNA. This helped us conducting a SAXS experiment taking into account the low resolution affinity and designed to provide the direct structural data of a complex of proteinaceous RNase P with a substrate tRNA.

For flawless translation of mRNA sequence into protein, tRNAs must undergo a series of essential maturation steps to be properly recognized and aminoacylated by aminoacyl-tRNA synthetase, and subsequently utilized by the ribosome. While all tRNAs carry a 30 -terminal CCA sequence that includes the site of aminoacylation, the additional 50 -G-1 position is a unique feature of most histidine tRNA species, serving as an identity element for the corresponding synthetase. In eukaryotes including yeast, both 30 -CCA and 50 -G-1 are added post-transcriptionally by tRNA nucleotidyltransferase and tRNAHis guanylyltransferase, respectively. Hence, it is possible that these two cytosolic enzymes compete for the same tRNA. Here, we investigate substrate preferences associated with CCA and G-1-addition to yeast cytosolic tRNAHis, which might result in a temporal order to these important processing events. We show that tRNA nucleotidyltransferase accepts tRNAHis transcripts independent of the presence of G-1; however, tRNAHis guanylyltransferase clearly prefers a substrate carrying a CCA terminus. Although many tRNA maturation steps can occur in a rather random order, our data demonstrate a likely pathway where CCA-addition precedes G-1 incorporation in S. cerevisiae. Evidently, the 30 -CCA triplet and a discriminator position A73 act as positive elements for G-1 incorporation, ensuring the fidelity of G-1 addition.