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Investigation of RNase P active site residues and catalytic domain interaction
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology. (Leif A. Kirsebom)ORCID iD: 0000-0001-5841-0991
2018 (English)Doctoral thesis, comprehensive summary (Other academic)
Description
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
Keyword [en]
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: urn:nbn:se:uu:diva-339607ISBN: 978-91-513-0214-0 (print)OAI: oai:DiVA.org:uu-339607DiVA: diva2:1176243
Public defence
2018-02-22, B/C4:305, Biomedical Center, Husargatan 3, Uppsala, 09:30 (English)
Opponent
Supervisors
Available from: 2018-01-31 Created: 2018-01-21 Last updated: 2018-01-31
List of papers
1. Impact of the P6- and P8/ P18-interactions in cleavage of model substrates by RNase P RNA with and without the specificity domain
Open this publication in new window or tab >>Impact of the P6- and P8/ P18-interactions in cleavage of model substrates by RNase P RNA with and without the specificity domain
(English)In: Article in journal (Refereed) Submitted
Abstract [en]

Abstract The natural trans-acting ribozyme RNase P RNA (RPR) is composed of two domains in which the catalytic (C-) domain mediates cleavage of various substrates. The C-domain alone, after removal of the second specificity (S-) domain, catalyzes this reaction as well, albeit with reduced efficiency. Here we provide experimental evidence indicating that efficient cleavage mediated by the Escherichia coli C-domain (Eco CP RPR) with and without the C5 protein likely depends on an interaction referred to as the "P6-mimic". Moreover, the P18 helix connects the C- and S-domains between its loop and the P8 helix in the S-domain (the P8/ P18-interaction). In contrast to the "P6-mimic", the presence of P18 does not contribute to the catalytic performance by the C-domain lacking the S-domain in cleavage of an all ribo model hairpin loop substrate while deletion or disruption of the P8/ P18-interaction in full-size RPR lowers the catalytic efficiency in cleavage of the same model hairpin loop substrate in keeping with previously reported data using precursor tRNAs. Consistent with that P18 is not required for cleavage mediated by the C-domain we show that the archaeal Pyrococcus furiosus RPR C-domain, which lacks the P18 helix, is catalytically active in trans without S-domain and any protein. Our data also suggest that the S-domain has a larger impact on catalysis for E. coli RPR compared to P. furiosus RPR. Finally, we provide data indicating that the absence8 of the S-domain and P18, or the P8/ P18-interaction in full-length RPR influences the charge distribution near the cleavage site in the RPR-substrate complex to a small but reproducible extent.

Place, publisher, year, edition, pages
Uppsala universitet
Keyword
RNase P/ Ribozyme/ Model substrates/ Divalent metal ions/ tRNA precursors/ RNA processing.
National Category
Biochemistry and Molecular Biology
Research subject
Biology with specialization in Molecular Biology
Identifiers
urn:nbn:se:uu:diva-339604 (URN)
Available from: 2018-01-21 Created: 2018-01-21 Last updated: 2018-01-22
2. Role of residue 248 in Escherichia coli RNase P RNA mediated cleavage
Open this publication in new window or tab >>Role of residue 248 in Escherichia coli RNase P RNA mediated cleavage
(English)Manuscript (preprint) (Other academic)
Abstract [en]

Abstract tRNA genes are transcribed as precursors and ribonuclease P (RNase P) generates the 5' end of tRNAs in the cell. It has been suggested that residue -1 (the residue immediately 5' of the scissile bond) in the substrate interacts with the well-conserved RNase P RNA (RPR) residue A248 (Escherichia coli numbering). The way A248 interacts with residue -1 is not clear. To gain insight into the role of A248, we analyzed cleavage as a function of A248 substitutions and N-1 nucleobase identity by using pre-tRNA and three all-ribo model substrates. Our findings 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. This leads to positioning of Mg2+ that activates the water that acts as the nucleophile resulting in efficient and correct cleavage. Furthermore, we propose that the role of A248 is to exclude bulk water from access to the amino acid acceptor stem, thereby preventing non-specific hydrolysis of the pre-tRNA. Finally, base stacking is discussed as a way to protect functionally important base-pairing interactions from non-specific hydrolysis, thereby ensuring high fidelity during RNA processing and the decoding of mRNA.

Keyword
RNase P/Ribozyme/Divalent metal ions/tRNA precursors/ tRNA processing.
National Category
Biochemistry and Molecular Biology
Research subject
Biology with specialization in Molecular Biology
Identifiers
urn:nbn:se:uu:diva-339605 (URN)
Available from: 2018-01-21 Created: 2018-01-21 Last updated: 2018-01-22
3. Cleavage of Model Substrates by Arabidopsis thaliana PRORP1 Reveals New Insights into Its Substrate Requirements
Open this publication in new window or tab >>Cleavage of Model Substrates by Arabidopsis thaliana PRORP1 Reveals New Insights into Its Substrate Requirements
Show others...
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.

National Category
Medicinal Chemistry
Identifiers
urn:nbn:se:uu:diva-307895 (URN)10.1371/journal.pone.0160246 (DOI)000381369500026 ()
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

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