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Cleavage of Model Substrates by Arabidopsis thaliana PRORP1 Reveals New Insights into Its Substrate Requirements
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Chemical Biology.
Ohio State Univ, Dept Chem & Biochem, Ctr RNA Biol, Columbus, OH 43210 USA..
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Chemical Biology. AstraZeneca R&D, Discovery Sci, Cambridge Sci Pk, Cambridge CB4 0WG, England..
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Chemical Biology.
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2016 (English)In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 11, no 8, article id e0160246Article in journal (Refereed) Published
Abstract [en]

Two broad classes of RNase P trim the 5' leader of precursor tRNAs (pre-tRNAs): ribonucleoprotein (RNP)- and proteinaceous (PRORP)-variants. These two RNase P types, which use different scaffolds for catalysis, reflect independent evolutionary paths. While the catalytic RNA-based RNP form is present in all three domains of life, the PRORP family is restricted to eukaryotes. To obtain insights on substrate recognition by PRORPs, we examined the 5' processing ability of recombinant Arabidopsis thaliana PRORP1 (AtPRORP1) using a panel of pre-tRNA(Ser) variants and model hairpin-loop derivatives (pATSer type) that consist of the acceptor-T-stem stack and the T-/D-loop. Our data indicate the importance of the identity of N-1 (the residue immediately 5' to the cleavage site) and the N-1: N+73 base pair for cleavage rate and site selection of pre-tRNA(Ser) and pATSer. The nucleobase preferences that we observed mirror the frequency of occurrence in the complete suite of organellar pre-tRNAs in eight algae/plants that we analyzed. The importance of the T-/D-loop in pre-tRNA(Ser) for tight binding to AtPRORP1 is indicated by the 200-fold weaker binding of pATSer compared to pre-tRNA(Ser), while the essentiality of the T-loop for cleavage is reflected by the near-complete loss of activity when a GAAA-tetraloop replaced the T-loop in pATSer. Substituting the 2'-OH at N-1 with 2'-H also resulted in no detectable cleavage, hinting at the possible role of this 2'-OH in coordinating Mg2+ ions critical for catalysis. Collectively, our results indicate similarities but also key differences in substrate recognition by the bacterial RNase P RNP and AtPRORP1: while both forms exploit the acceptor-T-stem stack and the elbow region in the pre-tRNA, the RNP form appears to require more recognition determinants for cleavage-site selection.

Place, publisher, year, edition, pages
2016. Vol. 11, no 8, article id e0160246
National Category
Medicinal Chemistry
Identifiers
URN: urn:nbn:se:uu:diva-307895DOI: 10.1371/journal.pone.0160246ISI: 000381369500026OAI: oai:DiVA.org:uu-307895DiVA, id: diva2:1048798
Funder
Swedish Research Council, Dnr 349-2006-267 Dnr 621-2011-5848Carl Tryggers foundation
Note

De två första författarna delar förstaförfattarskapet.

Available from: 2016-11-22 Created: 2016-11-22 Last updated: 2018-01-22Bibliographically approved
In thesis
1. Investigation of RNase P active site residues and catalytic domain interaction
Open this publication in new window or tab >>Investigation of RNase P active site residues and catalytic domain interaction
2018 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

RNase P is an essential endoribonuclease responsible for the maturation of the tRNA 5’end. The RNase P family encompasses the ribozyme based, RNase P RNP, and proteinaceous RNase P (PRORP). The ribozyme based RNase P is widely distributed in most species while PRORP has so far mainly been found in some eukaryotic cells.

The RNase P RNP contains one RNA subunit (RPR), which is the catalytic moiety, and one or more protein subunits. The structural topology of the RPR is crucial for RNase P RNP to correctly and efficiently maintain its function. The RPR is composed of domains such as the specificity (S) and catalytic (C) domains, and structural elements that connect these.

The objectives of my thesis were to study the importance of structural elements in the C-domain of the RPR with respect to substrate interaction and catalysis. Another objective was to study substrate interaction in PRORP-mediated catalysis, and to compare RNase P RNP- and PRORP-mediated cleavage. To achieve this I have studied cleavage of both pre-tRNA and model hairpin loop substrates with RPR variants carrying deletions and base substitutions, and PRORP1 from Arabidopsis thaliana.

My data provide evidence for an intra domain interaction, referred to as the P6-mimic, in the RPR C-domain. The P6-mimic forms when the S-domain of the RPR is deleted and it contributes to catalysis. The inter domain P8/P18 interaction, which connects the S- and the C-domains, plays an important role for catalysis. My data suggest that, in the absence of the S-domain, P18 does not contribute to catalysis raising the possibility that the P8/ P18-interaction acts as a structural mediator between the TSL/ TBS-interaction site in the S-domain and the active center that ensures correct and efficient cleavage. This is consistent with that RNase P RNP operates through an induced fit mechanism.  

Furthermore, on the basis of biochemical and genetic data the well-conserved A248 in the RPR has been proposed to form a cis Watson-Crick/Watson-Crick (cis WC/WC) pair with the residue immediately 5' of the cleavage site, N-1, in the substrate. My data does not support this cis WC/WC pairing. Rather, the data are consistent with a model where the structural topology of the active site varies and depends on the identity of the nucleobases at, and in proximity to, the cleavage site and their potential to interact. As a consequence, this affects the positioning of Mg2+ that activates the water that acts as the nucleophile resulting in efficient and correct cleavage. In this scenario it is suggested that the role of A248 is to exclude bulk water from accessing the amino acid acceptor stem and thereby prevent non-specific hydrolysis of the pre-tRNA. In a broader perspective, base stacking might be a way to prevent access of water to functionally important base pairing interactions, and thereby ensuring high fidelity during RNA processing and decoding of mRNA.

As for RNase P RNP, my studies on PRORP1 indicate the importance of the identity of N-1 and the N-1: N+73 base pair in the substrate for efficient and correct cleavage. Although, the data indicate similarities they also provide key differences in substrate recognition by RNase P RNP and PRORP1 where the RNP form appears to require more recognition determinants for cleavage site selection.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2018. p. 58
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 1623
Keywords
RNase P, Ribozyme, PRORP, Induced fit model, RNase P-pre-tRNA interaction
National Category
Biochemistry and Molecular Biology
Research subject
Biology with specialization in Molecular Biology
Identifiers
urn:nbn:se:uu:diva-339607 (URN)978-91-513-0214-0 (ISBN)
Public defence
2018-02-22, B/C4:305, Biomedical Center, Husargatan 3, Uppsala, 09:30 (English)
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Supervisors
Available from: 2018-01-31 Created: 2018-01-21 Last updated: 2018-05-21

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