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Cleavage of model substrates by archaeal RNase P: role of protein cofactors in cleavage-site selection
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, Microbiology.
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, Microbiology.
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2011 (English)In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 39, no 3, 1105-1116 p.Article in journal (Refereed) Published
Abstract [en]

RNase P is a catalytic ribonucleoprotein primarily involved in tRNA biogenesis. Archaeal RNase P comprises a catalytic RNase P RNA (RPR) and at least four protein cofactors (RPPs), which function as two binary complexes (POP5•RPP30 and RPP21• RPP29). Exploiting the ability to assemble a functional Pyrococcus furiosus (Pfu) RNase P in vitro, we examined the role of RPPs in influencing substrate recognition by the RPR. We first demonstrate that Pfu RPR, like its bacterial and eukaryal counterparts, cleaves model hairpin loop substrates albeit at rates 90- to 200-fold lower when compared with cleavage by bacterial RPR, highlighting the functionally comparable catalytic cores in bacterial and archaeal RPRs. By investigating cleavage-site selection exhibited by Pfu RPR (±RPPs) with various model substrates missing consensus-recognition elements, we determined substrate features whose recognition is facilitated by either POP5•RPP30 or RPP21•RPP29 (directly or indirectly via the RPR). Our results also revealed that Pfu RPR + RPP21•RPP29 displays substrate-recognition properties coinciding with those of the bacterial RPR-alone reaction rather than the Pfu RPR, and that this behaviour is attributable to structural differences in the substrate-specificity domains of bacterial and archaeal RPRs. Moreover, our data reveal a hierarchy in recognition elements that dictates cleavage-site selection by archaeal RNase P.

Place, publisher, year, edition, pages
2011. Vol. 39, no 3, 1105-1116 p.
National Category
Biochemistry and Molecular Biology
Identifiers
URN: urn:nbn:se:uu:diva-147838DOI: 10.1093/nar/gkq732ISI: 000287257500035PubMedID: 20935047OAI: oai:DiVA.org:uu-147838DiVA: diva2:400927
Available from: 2011-02-28 Created: 2011-02-28 Last updated: 2017-12-11Bibliographically 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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