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Functional Coupling between a Distal Interaction and the Cleavage Site in Bacterial RNase-P-RNA-Mediated Cleavage
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
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2011 (English)In: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 411, no 2, 384-396 p.Article in journal (Refereed) Published
Abstract [en]

Bacterial RNase P consists of one protein and one RNA [RNase P RNA (RPR)]. RPR can process tRNA precursors correctly in the absence of the protein. Here we have used model hairpin loop substrates corresponding to the acceptor, T-stem, and T-loop of a precursor tRNA to study the importance of the T-loop structure in RPR-alone reaction. T-stem/loop (TSL) interacts with a region in RPR [TSL binding site (TBS)], forming TSL/TBS interaction. Altering the T-loop structure affects both cleavage site selection and rate of cleavage at the correct site +1 and at the alternative site -1. The magnitude of variation depended on the structures of the T-loop and the TBS region, with as much as a 150-fold reduction in the rate of cleavage at +1. Interestingly, for one T-loop structure mutant, no difference in the rate at -1 was detected compared to cleavage of the substrate with an unchanged T-loop, indicating that, in this case, the altered T-loop structure primarily influences events required for efficient cleavage at the correct site +1. We also provide data supporting a functional link between a productive TSL/TBS interaction and events at the cleavage site. Collectively, our findings emphasize the interplay between separate regions upon formation of a productive RPR substrate that leads to efficient and accurate cleavage. These new data provide support for an induced-fit mechanism in bacterial RPR-mediated cleavage at the correct site +1.

Place, publisher, year, edition, pages
2011. Vol. 411, no 2, 384-396 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-158314DOI: 10.1016/j.jmb.2011.05.049ISI: 000293938400007OAI: oai:DiVA.org:uu-158314DiVA: diva2:439106
Available from: 2011-09-06 Created: 2011-09-06 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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