Substrate Recognition by Protein-only Ribonuclease Ps

Ribonuclease P;RNase P;PRORP;Biological Chemistry;Science;Biological Chemistry

Klemm, BradleyO ; ; Brien, Patrick ;

University of Michigan

关键词: Ribonuclease P;    RNase P;    PRORP;    Biological Chemistry;    Science;    Biological Chemistry;   

Others  :  https://deepblue.lib.umich.edu/bitstream/handle/2027.42/135873/klemmbp_1.pdf?sequence=1&isAllowed=y

瑞士|英语

来源: The Illinois Digital Environment for Access to Learning and Scholarship

【 摘 要 】

Transfer RNA (tRNA) is the adaptor molecule that translates a nucleic acid sequence from the genome into a protein sequence.tRNAs are transcribed as precursor molecules (pre-tRNAs) that have extra sequence on both the 5’ and 3’ ends that must be removed before the tRNA can be functional in translation. Ribonuclease Ps (RNase Ps) are metal-dependent endonucleases responsible for the 5’ end maturation of pre-tRNAs. All domains of life encode an RNA-dependent RNase P, with a conserved RNA component that is fully catalytic in Bacteria. Eukaryotes may also encode protein-only RNase Ps (PRORPs). The PRORPs from the land plant Arabidopsis thaliana are the only RNase Ps responsible for 5’ end maturation of pre-tRNAs in all three tRNA-encoding cellular compartments. Plant PRORPs use several structural features to recognize their substrates. The tRNA anti-codon is dispensable for activity, while the D-loop is required. The 5’ leader sequence beyond two nucleotides and the entire 3’ trailer sequence do not increase substrate affinity or significantly affect catalysis. The three differentially localized PRORPs encoded by plants do not have specificity for substrates based on the compartment of tRNA origin. PRORPs display an alternative 5’ end selection behavior in vitro. Substrates with the potential to form an extra base pair in the acceptor stem are miscleaved, but PRORPs re-process them to the correct 5’ end in vitro. Increasing ionic strength reduces the binding affinity and observed rate constants for Arabidopsis PRORPs, revealing 4-5 direct ionic contacts to backbone phosphates. The inhibition of catalysis reveals that the PRORP kinetic mechanism is more complicated than previously proposed. PRORPs contain pentatricopeptide repeat (PPR) RNA-binding domains, which have also been identified in single-stranded RNA binding proteins. A novel tRNA-recognition surface on the PRORP PPRs is conserved among PRORPs. Photo-activatable non-natural amino acid (NNAA) cross-linkers yield covalently bound PRORP-substrate for mapping interactions within the complex. We use a primer extension assay to identify the cross-linked sites on the pre-tRNA substrate. Overall, this work provides key insights into substrate recognition by an important class of endonucleases, including molecular determinants of substrate recognition, and develops a method to expand and continue these studies.