The RNR motif of <i>B. subtilis</i> RNase P protein interacts with both PRNA and pre-tRNA to stabilize an active conformer

Koutmou, Kristin S.; Day-Storms, Jeremy J.; Fierke, Carol A.

doi:10.1261/rna.2742511

Cited by 13 publications

(23 citation statements)

References 41 publications

(104 reference statements)

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“…Similar decreases in the conformational change rate constant (2- to 5-fold) have previously been observed for alanine mutations in conserved arginine residues in P protein. 76 We speculate that the bulged U51 nucleotide contributes to the conformational change step during the RNase P-catalyzed reaction.…”

Section: Resultsmentioning

confidence: 88%

Inner-Sphere Coordination of Divalent Metal Ion with Nucleobase in Catalytic RNA

2017

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Identification of the function of metal ions and the RNA moieties, particularly nucleobases, that bind metal ions are important questions in RNA catalysis. Here we combine single-atom and abasic substitutions to probe functions of conserved nucleobases in ribonuclease P (RNase P). Structural and biophysical studies of bacterial RNase P propose direct coordination of metal ions by the nucleobases of conserved uridine and guanosine in helix P4 of the RNA subunit (P RNA). To biochemically probe the function of metal ion interactions, we substituted the universally conserved bulged uridine (U51) in the P4 helix of circularly permuted Bacillus subtilis P RNA with 4-thiouridine, 4-deoxyuridine and abasic modifications and G378/379 with 2-aminopurine, N7-deazaguanosine, and 6-thioguanosine. The functional group modifications of U51 decrease RNase P-catalyzed phosphodiester bond cleavage 16- to 23-fold, as measured by the single-turnover cleavage rate constant. The activity of the 4-thiouridine RNase P is partially rescued by addition of Cd(II) or Mn(II) ions. This is the first time a metal-rescue experiment provides evidence for inner-sphere divalent metal ion coordination with a nucleobase. Modifications of G379 modestly decrease the cleavage activity of RNase P, suggesting outer-sphere coordination of O6 on G379 to a metal ion. These data provide biochemical evidence for catalytically important interactions of the P4 helix of P RNA with metal ions, demonstrating that the bulged uridine coordinates at least one catalytic metal ion through an inner-sphere interaction. The combination of single-atom and abasic nucleotide substitutions provides a powerful strategy to probe functions of conserved nucleobases in large RNAs.

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Section: Resultsmentioning

confidence: 88%

Inner-Sphere Coordination of Divalent Metal Ion with Nucleobase in Catalytic RNA

2017

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“…D and T-stem loops, 5′ leader length) based on in vitro kinetic experiments and the co-crystal structure of RNase P with tRNA Phe 21; 39; 40; 41; 42; 43; 44; 45; 46; 47; 48 . Activity assays and footprinting experiments indicate that PRORP1 recognizes individual residues in the pre-tRNA D- and T-loops 49 but it is unknown if, like RNA-based RNase P, the pre-tRNA 5′ leader and immature 3′ trailer length are also determinants of PRORP substrate recognition.…”

Section: Discussionmentioning

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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“…The protein component of the bacterial RNA-based RNase P is essential for catalysis in vivo but not in vitro at high salt (29). This protein interacts with both the pre-tRNA 5′ leader and the ribozyme to enhance substrate and metal ion affinity and to stabilize the RNA active site (29)(30)(31). In contrast, the proteinaceous RNase P has the ability to recognize, orient, and bind pre-tRNA and metal ions without any extraneous assistance.…”

Section: Discussionmentioning

confidence: 99%

Mitochondrial ribonuclease P structure provides insight into the evolution of catalytic strategies for precursor-tRNA 5′ processing

Howard¹,

Lim²,

Fierke

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

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Ribonuclease P (RNase P) catalyzes the maturation of the 5′ end of tRNA precursors. Typically these enzymes are ribonucleoproteins with a conserved RNA component responsible for catalysis. However, protein-only RNase P (PRORP) enzymes process precursor tRNAs in human mitochondria and in all tRNA-using compartments of Arabidopsis thaliana. PRORP enzymes are nuclear encoded and conserved among many eukaryotes, having evolved recently as yeast mitochondrial genomes encode an RNase P RNA. Here we report the crystal structure of PRORP1 from A. thaliana at 1.75 Å resolution, revealing a prototypical metallonuclease domain tethered to a pentatricopeptide repeat (PPR) domain by a structural zinc-binding domain. The metallonuclease domain is a unique high-resolution structure of a Nedd4-BP1, YacP Nucleases (NYN) domain that is a member of the PIN domain-like fold superfamily, including the FLAP nuclease family. The structural similarity between PRORP1 and the FLAP nuclease family suggests that they evolved from a common ancestor. Biochemical data reveal that conserved aspartate residues in PRORP1 are important for catalytic activity and metal binding and that the PPR domain also enhances activity, likely through an interaction with pre-tRNA. These results provide a foundation for understanding tRNA maturation in organelles. Furthermore, these studies allow for a molecular-level comparison of the catalytic strategies used by the only known naturally evolved protein and RNA-based catalysts that perform the same biological function, pre-tRNA maturation, thereby providing insight into the differences between the prebiotic RNA world and the present protein-dominated world.catalytic mechanism | magnesium | molecular recognition A ccording to the RNA world hypothesis, RNA played dual roles as carrier of genetic information and catalyst in a prebiotic world. However, over eons of evolution, proteins with their expanded 20-aa alphabet and greater structural and functional complexity took over many RNA-based functions. Structural insights into this evolutionary transition are limited because of the lack of examples of RNA and protein macromolecules that perform the same biological function in nature. One notable exception is ribonuclease P (RNase P) that catalyzes maturation of the 5′ end of tRNA across all domains of life. Until recently, all known RNase P enzymes included a catalytic RNA component. The discovery of a protein-only RNase P [proteinacous RNase P (PRORP)] from human mitochondria and Arabidopsis thaliana has dramatically shifted this paradigm (1-3). These enzymes represent a unique class of metallonucleases and are conserved among many eukaroytes (1, 2). A. thaliana encodes three PRORP enzymes (PRORP1, -2, and -3) that catalyze pretRNA processing (2). PRORP1 localizes to the mitochondria and chloroplast whereas PRORP2 and PRORP3 localize to the nucleus (2), suggesting that protein-based enzymes catalyze pretRNA maturation in these cellular locations (2, 3). To gain mechanistic and evolutionary insights into the PRORP...

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The RNR motif of B. subtilis RNase P protein interacts with both PRNA and pre-tRNA to stabilize an active conformer

Cited by 13 publications

References 41 publications

Inner-Sphere Coordination of Divalent Metal Ion with Nucleobase in Catalytic RNA

Inner-Sphere Coordination of Divalent Metal Ion with Nucleobase in Catalytic RNA

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

Mitochondrial ribonuclease P structure provides insight into the evolution of catalytic strategies for precursor-tRNA 5′ processing

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