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Distal to Proximal—Functional Coupling in RNase P RNA-mediated Catalysis
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology. (Leif A. Kirsebom)
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 [en]
Ribozyme, RNase P, Induced fit model, tRNA progressing, Substrate interaction
National Category
Biochemistry and Molecular Biology
Identifiers
URN: urn:nbn:se:uu:diva-159312ISBN: 978-91-554-8175-9 (print)OAI: oai:DiVA.org:uu-159312DiVA: diva2:444297
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
List of papers
1. Evidence for induced fit in bacterial RNase P RNA-mediated cleavage
Open this publication in new window or tab >>Evidence for induced fit in bacterial RNase P RNA-mediated cleavage
2007 (English)In: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 372, no 5, 1149-1164 p.Article in journal (Refereed) Published
Abstract [en]

RNase P with its catalytic RNA subunit is involved in the processing of a number of RNA precursors with different structures. However, precursor tRNAs are the most abundant substrates for RNase P. Available data suggest that a tRNA is folded into its characteristic structure already at the precursor state and that RNase P recognizes this structure. The tRNA D-/T-loop domain (TSL-region) is suggested to interact with the specificity domain of RNase P RNA while residues in the catalytic domain interact with the cleavage site. Here, we have studied the consequences of a productive interaction between the TSL-region and its binding site (TBS) in the specificity domain using tRNA precursors and various hairpin-loop model substrates. The different substrates were analyzed with respect to cleavage site recognition, ground-state binding, cleavage as a function of the concentration of Mg2+ and the rate of cleavage under conditions where chemistry is suggested to be rate limiting using wild-type Escherichia coli RNase P RNA, M1 RNA, and M1 RNA variants with structural changes in the TBS-region. On the basis of our data, we conclude that a productive TSL/TBS interaction results in a conformational change in the M1 RNA substrate complex that has an effect on catalysis. Moreover, it is likely that this conformational change comprises positioning of chemical groups (and Mg2+) at and in the vicinity of the cleavage site. Hence, our findings are consistent with an induced-fit mechanism in RNase P RNA-mediated cleavage.

Keyword
RNase P, ribozyme, divalent metal ions, tRNA precursors, tRNA processing
National Category
Biological Sciences
Identifiers
urn:nbn:se:uu:diva-14904 (URN)10.1016/j.jmb.2007.07.030 (DOI)000249817500003 ()17719605 (PubMedID)
Available from: 2008-01-31 Created: 2008-01-31 Last updated: 2017-12-11Bibliographically approved
2. Functional Coupling between a Distal Interaction and the Cleavage Site in Bacterial RNase-P-RNA-Mediated Cleavage
Open this publication in new window or tab >>Functional Coupling between a Distal Interaction and the Cleavage Site in Bacterial RNase-P-RNA-Mediated Cleavage
Show others...
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.

Keyword
RNase P, ribozyme, divalent metal ions, tRNA precursors, tRNA processing
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:uu:diva-158314 (URN)10.1016/j.jmb.2011.05.049 (DOI)000293938400007 ()
Available from: 2011-09-06 Created: 2011-09-06 Last updated: 2017-12-08Bibliographically approved
3. Cleavage of model substrates by archaeal RNase P: role of protein cofactors in cleavage-site selection
Open this publication in new window or tab >>Cleavage of model substrates by archaeal RNase P: role of protein cofactors in cleavage-site selection
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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.

National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:uu:diva-147838 (URN)10.1093/nar/gkq732 (DOI)000287257500035 ()20935047 (PubMedID)
Available from: 2011-02-28 Created: 2011-02-28 Last updated: 2017-12-11Bibliographically approved
4. Cleavage mediated by the catalytic domain of bacterial RNase P RNA
Open this publication in new window or tab >>Cleavage mediated by the catalytic domain of bacterial RNase P RNA
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.

Keyword
RNase P/Ribozyme/Divalent metal ions/tRNA precursors/tRNA processing
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:uu:diva-159311 (URN)10.1016/j.jmb.2012.05.020 (DOI)000308681000005 ()
Available from: 2011-09-27 Created: 2011-09-27 Last updated: 2017-12-08Bibliographically approved
5. The -1 residue and transition-state stabilization in RNase P RNA-mediated cleavage
Open this publication in new window or tab >>The -1 residue and transition-state stabilization in RNase P RNA-mediated cleavage
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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
RNase P RNA, -1 residue
National Category
Biochemistry and Molecular Biology
Research subject
Biology with specialization in Molecular Biology
Identifiers
urn:nbn:se:uu:diva-159310 (URN)
Available from: 2011-09-27 Created: 2011-09-27 Last updated: 2011-11-04

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