Poster abstracts

Poster number 154 submitted by Abigail Jackson

The N-1 nucleotide identity and discriminator base, along with conformational dynamics govern cleavage and binding by Arabidopsis thaliana PRORP2

Abigail Jackson (University of Michigan), Nickolas Manolias (University of Michigan), Markos Koutmos (University of Michigan)

Abstract:
RNase P is an endoribonuclease that carries out the first step of pre-tRNA maturation by cleaving the 5' leader sequence of pre-tRNAs. It belongs to one of the few naturally evolved RNA-cleaving chemistries, comprising two families—RNA-based and protein-based—that share substrate recognition strategies despite using fundamentally different active sites to cleave the bond between the N-1 and N+1 nucleotides. The PRORP family has diversified considerably, including human mitochondrial (mt) RNase P, single-subunit nuclear and organellar PRORPs, and the recently identified minimal PRORP, HARP. While single-subunit PRORPs have been structurally and biochemically characterized, the kinetics and thermodynamics governing recognition of native pre-tRNA substrates require further exploration. Here, we characterize substrate-dependent cleavage and binding by Arabidopsis thaliana (At) PRORP2, asking how the identity and spacing of the N-1 nucleotide and discriminator base, together with substrate stability, influence cleavage rate and binding affinity. Using gel-based and fluorescence polarization (FP) cleavage assays, FP binding assays, UV-Vis melting curves, and computational pre-tRNA modeling, we found that N-1 and discriminator base identity, and to a lesser extent their spacing, significantly influenced cleavage efficiency. We also found a strong negative correlation between substrate melting temperature and dissociation constant (KD, app), supported by pre-tRNA ensemble modeling. Together, these findings suggest that substrate structural stability, alongside sequence identity at the cleavage site, is an important determinant of substrate discrimination by At PRORP2. This adds to the evidence in ribonuclease enzymology that RNA recognition often depends on conformational and thermodynamic properties in addition to sequence alone.

References:
Gutmann, B.; Gobert, A.; Giegé, P. PRORP Proteins Support RNase P Activity in Both Organelles and the Nucleus in Arabidopsis. Genes Dev 2012, 26 (10), 1022–1027.

Pinker, F.; Bonnard, G.; Gobert, A.; Gutmann, B.; Hammani, K.; Sauter, C.; Gegenheimer, P. A.; Giegé, P. PPR Proteins Shed a New Light on RNase P Biology. RNA Biol 2013, 10 (9), 1457–1468.

Klemm, B. P.; Wu, N.; Chen, Y.; Liu, X.; Kaitany, K. J.; Howard, M. J.; Fierke, C. A. The Diversity of Ribonuclease P: Protein and RNA Catalysts With Analogous Biological Functions. Biomolecules 2016, 6 (2), 27.

Keywords: tRNA maturation, RNase P, tRNA 5 cleavage