Mechanistic Studies Reveal Similar Catalytic Strategies for Phosphodiester Bond Hydrolysis by Protein-only and RNA-dependent Ribonuclease P

Howard, Michael J.; Klemm, Bradley P.; Fierke, Carol A.

doi:10.1074/jbc.m115.644831

Cited by 36 publications

(77 citation statements)

References 48 publications

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“…The observed rate constant under these conditions is proposed to mainly reflect the chemical cleavage step (Howard et al 2015). In all cases, the leader length was consistent with cleavage at the correct site.…”

Section: Prorp1 Substrate Recognition Has Little Dependence On Leadermentioning

confidence: 71%

“…To determine whether PRORP1 shares a similar dependence on leader length, we measured the binding affinity and single-turnover (STO) cleavage rate constant for B. subtilis pre-tRNA Asp lacking the 3 ′ -CCA sequence. This substrate was previously used to interrogate both the mechanism of PRORP (Howard et al 2015) and leader interactions with B. subtilis RNase P Rueda et al 2005;Hsieh and Fierke 2009). This allows a direct comparison between the recognition modes of PRORP1 and bacterial RNase P.…”

Section: Prorp1 Substrate Recognition Has Little Dependence On Leadermentioning

confidence: 99%

“…The central domain binds a Zn 2+ ion and structurally links the PPR and metallonuclease domains. The NYN-metallonuclease domain catalyzes phosphodiester bond hydrolysis and contains four conserved aspartates important for binding catalytic metal ions and catalysis (Howard et al 2012(Howard et al , 2015. Both the protein and RNA-based RNase P enzymes are proposed to use a two-metal ion mechanism for catalysis (Steitz and Steitz 1993;Howard et al 2015).…”

Section: Introductionmentioning

confidence: 99%

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Differential substrate recognition by isozymes of plant protein-only Ribonuclease P

Howard

Karasik

Klemm

et al. 2016

RNA

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Ribonuclease P (RNase P) catalyzes the cleavage of leader sequences from precursor tRNA (pre-tRNA). Typically, these enzymes are ribonucleic protein complexes that are found in all domains of life. However, a new class of RNase P has been discovered that is composed entirely of protein, termed protein-only RNase P (PRORP). To investigate the molecular determinants of PRORP substrate recognition, we measured the binding affinities and cleavage kinetics of Arabidopsis PRORP1 for varied pre-tRNA substrates. This analysis revealed that PRORP1 does not make significant contacts within the trailer or beyond N −1 of the leader, indicating that this enzyme recognizes primarily the tRNA body. To determine the extent to which sequence variation within the tRNA body modulates substrate selectivity and to provide insight into the evolution and function of PRORP enzymes, we measured the reactivity of the three Arabidopsis PRORP isozymes (PRORP1-3) with four pre-tRNA substrates. A 13-fold range in catalytic efficiencies (10 4 -10 5 M −1 s −1 ) was observed, demonstrating moderate selectivity for pre-tRNA substrates. Although PRORPs bind the different pre-tRNA species with affinities varying by as much as 100-fold, the three isozymes have similar affinities for a given pre-tRNA, suggesting similar binding modes. However, PRORP isozymes have varying degrees of cleavage fidelity, which is dependent on the pre-tRNA species and the presence of a 3 ′ -discriminator base. This work defines molecular determinants of PRORP substrate recognition that provides insight into this new class of RNA processing enzymes.

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Section: Prorp1 Substrate Recognition Has Little Dependence On Leadermentioning

confidence: 71%

Section: Prorp1 Substrate Recognition Has Little Dependence On Leadermentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

Differential substrate recognition by isozymes of plant protein-only Ribonuclease P

Howard

Karasik

Klemm

et al. 2016

RNA

Self Cite

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“…Thus far, PRORP1 is the best-characterized PRORP from any organism and the only PRORP for which there is a crystal structure where all of its domains are intact 8; 10; 11 . A recent study evaluating the in vitro pH profile and metal dependence of PRORP1 cleavage activity proposed that, like the “ribozyme-based” RNase P, PRORP1 uses a metal-bound water nucleophile to cleave pre-tRNA substrates 12 . The precise molecular recognition strategies used by PRORPs remain largely unknown.…”

Section: Introductionmentioning

confidence: 99%

“…While the investigations of PRORP1 provide an important first step towards understanding how PRORPs work 8; 12; 15; 16 the study of a single enzyme cannot reveal how PRORPs generally function; comparison of the structure and function of multiple proteins is necessary to understand any given class of enzymes. Here, we present the first x-ray structure and biochemical characterization of a nuclear localized PRORP, A. thaliana PRORP2.…”

Section: Introductionmentioning

confidence: 99%

Nuclear Protein-Only Ribonuclease P2 Structure and Biochemical Characterization Provide Insight into the Conserved Properties of tRNA 5′ End Processing Enzymes

Karasik

Shanmuganathan

Howard

et al. 2016

Journal of Molecular Biology

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Protein-only RNase Ps (PRORPs) are a recently discovered class of RNA processing enzymes that catalyze maturation of the 5′ end of precursor tRNAs (pre-tRNAs) in Eukaryotes. PRORPs are found in the nucleus and/or organelles of most eukaryotic organisms. Arabidopsis thaliana is a representative organism that contains PRORP enzymes (PRORP1, PRORP2, PRORP3) in both its nucleus and organelles; PRORP2 and 3 localize to the nucleus, PRORP1 to the chloroplast and the mitochondria. Apart from their identification, almost nothing is known about the structure and function of PRORPs that act in the nucleus. Here we use a combination of biochemical assays and X-ray crystallography to characterize A. thaliana PRORP2. We solved the crystal structure of PRORP2 (3.2 Å) revealing an overall V-shaped protein and conserved metallonuclease active site structure. Our biochemical studies indicate that PRORP2 requires Mg2+ for catalysis and catalyzes the maturation of nuclear encoded substrates up to 10-fold faster than mitochondrial encoded precursor nad6 t-element under single turnover conditions. We also demonstrate that PRORP2 preferentially binds pre-tRNAs containing short 5′ leaders and 3′ trailers; however, leader and trailer length do not significantly alter the observed rate constants of PRORP2 in single turnover cleavage assays. Our data provide a biochemical and structural framework to begin understanding how nuclear localized PRORPs recognize and cleave their substrates.

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Multiple structural flavors of RNase P in precursor tRNA processing

Sridhara

2024

WIREs RNA

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The precursor transfer RNAs (pre‐tRNAs) require extensive processing to generate mature tRNAs possessing proper fold, structural stability, and functionality required to sustain cellular viability. The road to tRNA maturation follows an ordered process: 5′‐processing, 3′‐processing, modifications at specific sites, if any, and 3′‐CCA addition before aminoacylation and recruitment to the cellular protein synthesis machinery. Ribonuclease P (RNase P) is a universally conserved endonuclease in all domains of life, performing the hydrolysis of pre‐tRNA sequences at the 5′ end by the removal of phosphodiester linkages between nucleotides at position −1 and +1. Except for an archaeal species: Nanoarchaeum equitans where tRNAs are transcribed from leaderless‐position +1, RNase P is indispensable for life and displays fundamental variations in terms of enzyme subunit composition, mechanism of substrate recognition and active site architecture, utilizing in all cases a two metal ion‐mediated conserved catalytic reaction. While the canonical RNA‐based ribonucleoprotein RNase P has been well‐known to occur in bacteria, archaea, and eukaryotes, the occurrence of RNA‐free protein‐only RNase P in eukaryotes and RNA‐free homologs of Aquifex RNase P in prokaryotes has been discovered more recently. This review aims to provide a comprehensive overview of structural diversity displayed by various RNA‐based and RNA‐free RNase P holoenzymes towards harnessing critical RNA–protein and protein–protein interactions in achieving conserved pre‐tRNA processing functionality. Furthermore, alternate roles and functional interchangeability of RNase P are discussed in the context of its employability in several clinical and biotechnological applications.This article is categorized under: RNA Processing > tRNA Processing RNA Evolution and Genomics > RNA and Ribonucleoprotein Evolution RNA Interactions with Proteins and Other Molecules > RNA‐Protein Complexes

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Mechanistic Studies Reveal Similar Catalytic Strategies for Phosphodiester Bond Hydrolysis by Protein-only and RNA-dependent Ribonuclease P

Cited by 36 publications

References 48 publications

Differential substrate recognition by isozymes of plant protein-only Ribonuclease P

Differential substrate recognition by isozymes of plant protein-only Ribonuclease P

Nuclear Protein-Only Ribonuclease P2 Structure and Biochemical Characterization Provide Insight into the Conserved Properties of tRNA 5′ End Processing Enzymes

Multiple structural flavors of RNase P in precursor tRNA processing

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