Abstract
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 protein-only RNase P (PRORP), 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 in solution as well as that of its complex with a tRNA precursor by small-angle X-ray scattering. 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.
Highlights
RNase P is a universal enzyme that removes 5 leader sequences from tRNA precursors
The N-terminal arm consists of a pentatricopeptide repeat (PPR) domain made of five PPR motifs, the C-terminal arm consists of the NYN metallonuclease domain, and a bipartite zincbinding domain (ZBD) bridges the two arms together
Initial investigations of protein-only RNase P (PRORP) mode of action showed which tRNA residues are in contact with PRORP [11], but the way tRNA recognition is achieved by PRORP was unknown, and no data were available on the structure of the PRORP–tRNA complex and on the dynamics of the enzyme
Summary
RNase P is a universal enzyme that removes 5 leader sequences from tRNA precursors. The enzyme is essential for maturation of functional tRNAs and mRNA translation. The identification of tRNA residues in contact with PRORP [11] and RNP RNase P [19] has suggested that RNPs and PRORP use a similar strategy to recognize their substrates [11] Still, how this is achieved at the protein level remained unknown, and the dynamics required for PRORP mode of action was unexplored [20]. How this is achieved at the protein level remained unknown, and the dynamics required for PRORP mode of action was unexplored [20] To tackle these questions, we determined the crystal structure of Arabidopsis nuclear PRORP2 together with a SAXS model in solution and explored the role of its PPR domain for substrate binding. Our results identify structural features in PRORP2 important for the RNA binding process and suggest that the enzyme is flexible and undergoes conformational changes to perform its activity
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