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Cleavage mediated by the catalytic domain of bacterial RNase P RNA
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology. (Leif A. Kirsebom)
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology. (Leif A. Kirsebom)
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
2012 (English)In: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 422, no 2, 204-214 p.Article in journal (Refereed) Published
Abstract [en]

As for other RNA molecules RNase P RNA (RPR) is composed of domains and these have different functions. Here we provide data demonstrating that the catalytic (C) domain of Escherichia coli (Eco) RPR when separated from the specificity (S) domain mediates cleavage using various model hairpin loop substrates. Compared to full-size Eco RPR the rate of cleavage for the truncated RPR (CP RPR) was reduced 30- to 13000-fold. We provide data  that the magnitude of reduction in rate is substrate dependent and that the structural architecture of the -1/+73 plays a significant role where a C-1/G+73 pair had the most dramatic effect on the rate. Substitution of A248 (E. coli numbering), which is positioned near the cleavage site in the RNase P-substrate complex, with G in the CP RPR resulted in 30-fold rate improvement while strengthening the interaction between the RPR and the 3' end of the substrate only had a modest effect. Interestingly, while deleting the S-domain gave a reduction in the rate it resulted in a less erroneous RPR with respect to cleavage site selection. These data will be dicussed in view of our current understanding of the coupling between the distal interaction between the S-domain and events at the active site and in an evolutionary perspective.

Place, publisher, year, edition, pages
2012. Vol. 422, no 2, 204-214 p.
Keyword [en]
RNase P/Ribozyme/Divalent metal ions/tRNA precursors/tRNA processing
National Category
Biochemistry and Molecular Biology
Identifiers
URN: urn:nbn:se:uu:diva-159311DOI: 10.1016/j.jmb.2012.05.020ISI: 000308681000005OAI: oai:DiVA.org:uu-159311DiVA: diva2:444087
Available from: 2011-09-27 Created: 2011-09-27 Last updated: 2017-12-08Bibliographically approved
In thesis
1. Distal to Proximal—Functional Coupling in RNase P RNA-mediated Catalysis
Open this publication in new window or tab >>Distal to Proximal—Functional Coupling in RNase P RNA-mediated Catalysis
2011 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

RNase P is a ubiquitous ribonuclease responsible for removing the 5’ leader of tRNA precursor. Bacterial RNase P contains one RNA (RPR) and one protein (RPP) subunit. However, the number of protein variants depends on the origin. The RNA subunit is the catalytic subunit that in vitro cleaves its substrate with and without the protein subunit. Therefore RNase P is a ribozyme. However, the protein subunit is indispensable in vivo.

The objective of this thesis was to understand the mechanism of and substrate interaction in RPR-mediated cleavage, in particular the contributions of the two domains of RPR and the roles of the base at the -1 residue in the substrate. As model systems I have used bacterial (Eco) and archaeal (Pfu) RPRs.

The TSL (T-stem-loop) region of a tRNA precursor and the TBS (TSL-binding site) in the RPR S-domain interact upon RPR-substrate complex conformation. A productive TSL/TBS-interaction affects events at the cleavage site by influencing the positioning of chemical groups and/ or Mg2+ such that efficient and correct cleavage occurs consistent with an induced fit mechanism. With respect to events at the cleavage site, my data show that the identity of the residue immediately upstream the 5’ of the cleavage site (at -1) plays a significant role for efficient and accurate cleavage although its presence is not essential. My data also show that the RPR C-domain can cleave without the S-domain. However, the presence of the S-domain increases the efficiency of cleavage but lowers the accuracy. The structure of the S-domain of Pfu RPR differs from that of Eco RPR and my data suggest that the Pfu S-domain does not affect the accuracy in the same way as for Eco RPR. It also appears that the proteins that bind to the Pfu S-domain play a role in formation of a productive TSL/TBS-interaction. It is therefore possible that the proteins of Pfu RNase P have evolved to take over the role of the S-domain with respect to the interaction with the TSL-region of the substrate.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2011. 64 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 861
Keyword
Ribozyme, RNase P, Induced fit model, tRNA progressing, Substrate interaction
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:uu:diva-159312 (URN)978-91-554-8175-9 (ISBN)
Public defence
2011-11-11, B42, Bio mediacal Center (BMC), Husargatan 3, Uppsala, 10:15 (English)
Opponent
Supervisors
Available from: 2011-10-20 Created: 2011-09-27 Last updated: 2011-11-04Bibliographically approved

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Wu, ShiyingKirsebom, Leif A.

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