PMID:10843849
| Citation |
Busch, S, Kirsebom, LA, Notbohm, H and Hartmann, RK (2000) Differential role of the intermolecular base-pairs G292-C(75) and G293-C(74) in the reaction catalyzed by Escherichia coli RNase P RNA. J. Mol. Biol. 299:941-51 |
|---|---|
| Abstract |
We present a systematic investigation of the thermodynamic and kinetic role of the intermolecular G292-C(75 )and G293-C(74 )Watson-Crick base-pairs in the reaction catalyzed by Escherichia coli RNase P RNA. Single turnover kinetics were analyzed for wild-type RNase P RNA and two variants with a single G to C exchange (C292 or C293), either acting on wild-type precursor tRNA (ptRNA) or derivatives carrying a complementary change at the tRNA 3'-end (G(74)CA or CG(75)A). Ground state binding of tRNA was studied using three different methods, including a novel fluorescence-based assay measuring equilibrium binding. We conclude that: (1) the role of the G293-C(74 )interaction is essentially confined to Watson-Crick base-pairing, with no indication for crucial tertiary contacts involving this base-pair; (2) the G293-C(74 )pair, although being as important for ptRNA ground state binding as G292-C(75), is much less crucial to catalytic performance than the G292-C(75) pair; (3) disruption of the G292-C(75 )base-pair results in preferential destabilization of enzyme transition-state complexes; and (4) the identity of the G292-C(75) pair, as part of the higher-order structural context consisting of coplanar G292-C(75)-A258 and G291-G259-A(76 )triples, contributes to high affinity binding of ptRNA and catalytic efficiency. |
| Links |
PubMed Online version:10.1006/jmbi.2000.3789 |
| Keywords |
Base Pairing/genetics; Base Sequence; Binding, Competitive; Catalysis; Circular Dichroism; Endoribonucleases/chemistry; Endoribonucleases/genetics; Endoribonucleases/metabolism; Escherichia coli/enzymology; Escherichia coli/genetics; Escherichia coli Proteins; Fluorescein/metabolism; Fluorescence; Kinetics; Molecular Sequence Data; Mutation/genetics; Nucleic Acid Denaturation; RNA Precursors/chemistry; RNA Precursors/genetics; RNA Precursors/metabolism; RNA, Bacterial/chemistry; RNA, Bacterial/genetics; RNA, Bacterial/metabolism; RNA, Catalytic/chemistry; RNA, Catalytic/genetics; RNA, Catalytic/metabolism; RNA, Transfer, Gly/chemistry; RNA, Transfer, Gly/genetics; RNA, Transfer, Gly/metabolism; Ribonuclease P; Temperature; Thermodynamics |
| edit table |
Significance
You can help EcoliWiki by summarizing why this paper is useful
Useful Materials and Methods
You can help Ecoliwiki by describing the useful materials (strains, plasmids, antibodies, etc) described in this paper.
Annotations
<annotationlinks/>
EcoliWiki Links
Add links to pages that link here (e.g. gene, product, method pages)
References
See Help:References for how to manage references in EcoliWiki.