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

Chen, Tien‐Hao; Tanimoto, Akiko; Shkriabai, Nikoloz; Kvaratskhelia, Mamuka; Wysocki, Vicki H.; Gopalan, Venkat

doi:10.1093/nar/gkw391

Cited by 15 publications

(17 citation statements)

References 60 publications

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“…This is probably because the mutation at position 35 introduces a negative charge that can interfere with RNA binding. Still, although PPR1 mutant does not display a loss of RNase P activity, our data suggest that the PPR1 motif is in the vicinity of the RNA in accordance with previous work showing contacts between the N-terminal extremity of PRORP and tRNA (28). In contrast, PPR motifs 4 and 5 do not seem to be involved in RNA interaction because their mutations do not alter RNA binding and cleavage.…”

Section: Biophysical Analysis Of Prorp2-trna Complexsupporting

confidence: 90%

Biophysical analysis of Arabidopsis protein-only RNase P alone and in complex with tRNA provides a refined model of tRNA binding

Pinker

Schelcher

Fernández-Millán

et al. 2017

Journal of Biological Chemistry

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RNase P is a universal enzyme that removes 5' leader sequences from tRNA precursors. The enzyme is therefore essential for maturation of functional tRNAs and mRNA translation. RNase P represents a unique example of an enzyme that can occur either as ribonucleoprotein or as protein alone. The latter form of the enzyme, called protein-only RNase P (PRORP), is widespread in eukaryotes in which it can provide organellar or nuclear RNase P activities. Here, we have focused on nuclear PRORP2 and its interaction with tRNA substrates. Affinity measurements helped assess the respective importance of individual pentatricopeptide repeat motifs in PRORP2 for RNA binding. We characterized the PRORP2 structure by X-ray crystallography and by small-angle X-ray scattering in solution as well as that of its complex with a tRNA precursor by small-angle X-ray scattering. Of note, our study reports the first structural data of a PRORP-tRNA complex. Combined with complementary biochemical and biophysical analyses, our structural data suggest that PRORP2 undergoes conformational changes to accommodate its substrate. In particular, the catalytic domain and the RNA-binding domain can move around a central hinge. Altogether, this work provides a refined model of the PRORP-tRNA complex that illustrates how protein-only RNase P enzymes specifically bind tRNA and highlights the contribution of protein dynamics to achieve this specific interaction.

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Section: Biophysical Analysis Of Prorp2-trna Complexsupporting

confidence: 90%

Biophysical analysis of Arabidopsis protein-only RNase P alone and in complex with tRNA provides a refined model of tRNA binding

Pinker

Schelcher

Fernández-Millán

et al. 2017

Journal of Biological Chemistry

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“…Results thus represent indirect evidence to show the importance of the PPR domain for RNA recognition and do not conclusively show if and/or how PPR motifs confer specificity to PRORP enzymes. In another study, lysine residues positioned at both tips of PRORP Λ shape were found in contact with the pre-tRNA substrate [ 59 ], thus, in agreement with the general substrate binding process proposed for PRORP proteins [ 5 ]. Mutated residues that were shown to interact with pre-tRNA and/or to be essential for RNase P activity are shown in Figure 2 .…”

Section: Mechanistic Analyses Of Protein-only Rnase P Activitysupporting

confidence: 77%

Mechanistic and Structural Studies of Protein-Only RNase P Compared to Ribonucleoproteins Reveal the Two Faces of the Same Enzymatic Activity

2016

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RNase P, the essential activity that performs the 5′ maturation of tRNA precursors, can be achieved either by ribonucleoproteins containing a ribozyme present in the three domains of life or by protein-only enzymes called protein-only RNase P (PRORP) that occur in eukaryote nuclei and organelles. A fast growing list of studies has investigated three-dimensional structures and mode of action of PRORP proteins. Results suggest that similar to ribozymes, PRORP proteins have two main domains. A clear functional analogy can be drawn between the specificity domain of the RNase P ribozyme and PRORP pentatricopeptide repeat domain, and between the ribozyme catalytic domain and PRORP N4BP1, YacP-like Nuclease domain. Moreover, both types of enzymes appear to dock with the acceptor arm of tRNA precursors and make specific contacts with the corner of pre-tRNAs. While some clear differences can still be delineated between PRORP and ribonucleoprotein (RNP) RNase P, the two types of enzymes seem to use, fundamentally, the same catalytic mechanism involving two metal ions. The occurrence of PRORP and RNP RNase P represents a remarkable example of convergent evolution. It might be the unique witness of an ongoing replacement of catalytic RNAs by proteins for enzymatic activities.

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“…[4, 19] DNA templates containing a class III Φ6.5 promoter and the genes for either MR1 or MR2 model RNAs were generated using a pair of DNA oligonucleotides for each RNA (Table S2; Sigma). These oligonucleotides were designed to be complementary at their respective 3’-termini; the 17-bp complement allowed fill-in with Phusion DNA Polymerase (NEB).…”

Section: Methodsmentioning

confidence: 99%

An RNase P‐Based Assay for Accurate Determination of the 5′‐Deoxy‐5′‐azidoguanosine‐Modified Fraction of in Vitro‐Transcribed RNAs

et al. 2018

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Chemoenzymatic approaches are important for generating site-specific, chemically modified RNAs, a cornerstone for RNA structure-function correlation studies. T7 RNA polymerase (T7RNAP)-mediated in vitro transcription (IVT) of a DNA template containing the G-initiating class III Φ6.5 promoter is typically used to generate 5'-chemically modified RNAs by including a guanosine analogue (G analogue) initiator in the IVT. However, the yield of 5'-G analogue-initiated RNA is often poor and variable due to the high ratios of G analogue:GTP used in IVT. We recently reported that a T7RNAP P266L mutant afforded an approximately three-fold increase in fluorescent 5'-thienoguanosine-initiated pre-tRNA compared to the wild type T7RNAP. We have further explored the use of T7RNAP P266L to generate 5'-deoxy-5'-azidoguanosine ( G)-initiated RNA and found that the mutant yielded approximately four times more G-initiated pre-tRNA than the wild type in an IVT containing a 10:1 ratio of G:GTP. For accurate quantitation of the 5'- G-initiated RNA fraction, we employed RNase P, an endonuclease that catalyzes the removal of the 5'-leader in pre-tRNAs. Importantly, we show herein how RNase P can be leveraged for assessing 5'-G analogue incorporation in any RNA by rendering the target RNA, upon its binding to a customized external guide sequence RNA, into an unnatural substrate of RNase P. Such an approach in conjunction with T7RNAP P266L-based IVT should aid chemoenzymatic methods that are designed to generate 5'-chemically modified RNAs.

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

Cited by 15 publications

References 60 publications

Biophysical analysis of Arabidopsis protein-only RNase P alone and in complex with tRNA provides a refined model of tRNA binding

Biophysical analysis of Arabidopsis protein-only RNase P alone and in complex with tRNA provides a refined model of tRNA binding

Mechanistic and Structural Studies of Protein-Only RNase P Compared to Ribonucleoproteins Reveal the Two Faces of the Same Enzymatic Activity

An RNase P‐Based Assay for Accurate Determination of the 5′‐Deoxy‐5′‐azidoguanosine‐Modified Fraction of in Vitro‐Transcribed RNAs

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