Dataset Information

Use of chemical modification and mass spectrometry to identify substrate-contacting sites in proteinaceous RNase P, a tRNA processing enzyme.

ABSTRACT: Among all enzymes in nature, RNase P is unique in that it can use either an RNA- or a protein-based active site for its function: catalyzing cleavage of the 5'-leader from precursor tRNAs (pre-tRNAs). The well-studied catalytic RNase P RNA uses a specificity module to recognize the pre-tRNA and a catalytic module to perform cleavage. Similarly, the recently discovered proteinaceous RNase P (PRORP) possesses two domains - pentatricopeptide repeat (PPR) and metallonuclease (NYN) - that are present in some other RNA processing factors. Here, we combined chemical modification of lysines and multiple-reaction monitoring mass spectrometry to identify putative substrate-contacting residues in Arabidopsis thaliana PRORP1 (AtPRORP1), and subsequently validated these candidate sites by site-directed mutagenesis. Using biochemical studies to characterize the wild-type (WT) and mutant derivatives, we found that AtPRORP1 exploits specific lysines strategically positioned at the tips of it's V-shaped arms, in the first PPR motif and in the NYN domain proximal to the catalytic center, to bind and cleave pre-tRNA. Our results confirm that the protein- and RNA-based forms of RNase P have distinct modules for substrate recognition and cleavage, an unanticipated parallel in their mode of action.

SUBMITTER: Chen TH

PROVIDER: S-EPMC4914120 | biostudies-literature | 2016 Jun

REPOSITORIES: biostudies-literature

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Use of chemical modification and mass spectrometry to identify substrate-contacting sites in proteinaceous RNase P, a tRNA processing enzyme.

Chen Tien-Hao TH Tanimoto Akiko A Shkriabai Nikoloz N Kvaratskhelia Mamuka M Wysocki Vicki V Gopalan Venkat V

Nucleic acids research 20160510 11

Among all enzymes in nature, RNase P is unique in that it can use either an RNA- or a protein-based active site for its function: catalyzing cleavage of the 5'-leader from precursor tRNAs (pre-tRNAs). The well-studied catalytic RNase P RNA uses a specificity module to recognize the pre-tRNA and a catalytic module to perform cleavage. Similarly, the recently discovered proteinaceous RNase P (PRORP) possesses two domains - pentatricopeptide repeat (PPR) and metallonuclease (NYN) - that are present ...[more]

PMID: 27166372

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Project description:RNase Z is a widely distributed and usually essential endoribonuclease involved in the 3'-end maturation of transfer RNAs (tRNAs). A CCA triplet that is needed for tRNA aminoacylation in protein translation is added by a nucleotidyl-transferase after the 3'-end processing by RNase Z. However, a considerable proportion of the archaeal pre-tRNAs genetically encode a CCA motif, while the enzymatic characteristics of the archaeal RNase (aRNase) Zs in processing CCA-containing pre-tRNAs remain unclear. This study intensively characterized two methanomicrobial aRNase Zs, the Methanolobus psychrophilus mpy-RNase Z and the Methanococcus maripaludis mmp-RNase Z, particularly focusing on the properties of processing the CCA-containing pre-tRNAs, and in parallel comparison with a bacterial bsu-RNase Z from Bacillus subtilis. Kinetic analysis found that Co2+ supplementation enhanced the cleavage efficiency of mpy-RNase Z, mmp-RNase Z, and bsu-RNase Z for 1400-, 2990-, and 34-fold, respectively, and Co2+ is even more indispensable to the aRNase Zs than to bsu-RNase Z. Mg2+ also elevated the initial cleavage velocity (V0) of bsu-RNase Z for 60.5-fold. The two aRNase Zs exhibited indiscriminate efficiencies in processing CCA-containing vs. CCA-less pre-tRNAs. However, V0 of bsu-RNase Z was markedly reduced for 1520-fold by the CCA motif present in pre-tRNAs under Mg2+ supplementation, but only 5.8-fold reduced under Co2+ supplementation, suggesting Co2+ could ameliorate the CCA motif inhibition on bsu-RNase Z. By 3'-RACE, we determined that the aRNase Zs cleaved just downstream the discriminator nucleotide and the CCA triplet in CCA-less and CCA-containing pre-tRNAs, thus exposing the 3'-end for linking CCA and the genetically encoded CCA triplet, respectively. The aRNase Zs, but not bsu-RNase Z, were also able to process the intron-embedded archaeal pre-tRNAs, and even process pre-tRNAs that lack the D, T, or anticodon arm, but strictly required the acceptor stem. In summary, the two methanomicrobial aRNase Zs use cobalt as a metal ligand and process a broad spectrum of pre-tRNAs, and the characteristics would extend our understandings on aRNase Zs.

Dataset Information

Use of chemical modification and mass spectrometry to identify substrate-contacting sites in proteinaceous RNase P, a tRNA processing enzyme.

Publications

Use of chemical modification and mass spectrometry to identify substrate-contacting sites in proteinaceous RNase P, a tRNA processing enzyme.

Similar Datasets

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