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The -1 residue and transition-state stabilization in RNase P RNA-mediated cleavage
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)
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(English)Manuscript (preprint) (Other academic)
Abstract [en]

We have used model substrates carrying modified nucleotides at the site immediately 5' of the scissile bond, the -1 position, to get a detailed understanding of RNase P RNA mediated cleavage. We show that the base at -1 is not essential but its presence and identity contributes to efficiency, correctness of cleavage and stabilization of the transitions state by 5.1 kcal. When U or C is present at -1, the carbonyl oxygen at position 2 contributes by 2.6 kcal of the 5.1 kcal and thus acts as a positive factor. For substrates with purines at -1, an exocyclic amine at position 2 has as a negative impact on cleavage at the canonical site. We will discuss our findings in view of a model where RNase P cleavage proceeds through a conformational-assisted mechanism that positions the metal-activated H2O for an in-line attack on the phosphorous atom that leads to breakage of the phosphodiester bond.

Keyword [en]
RNase P RNA, -1 residue
National Category
Biochemistry and Molecular Biology
Research subject
Biology with specialization in Molecular Biology
Identifiers
URN: urn:nbn:se:uu:diva-159310OAI: oai:DiVA.org:uu-159310DiVA: diva2:444081
Available from: 2011-09-27 Created: 2011-09-27 Last updated: 2011-11-04
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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