Differential role of the intermolecular base-pairs G292-C(75) and G293-C(74) in the reaction catalyzed by Escherichia coli RNase P RNA.
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.
Busch
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Medizinische Universität zu Lübeck, Institut für Biochemie, Ratzeburger Allee 160, Lübeck, D-23538, Germany.
Kirsebom
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LA
Notbohm
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Hartmann
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RK
D020029:Base Pairing / Q000235:genetics*; D001483:Base Sequence; D001667:Binding, Competitive; D002384:Catalysis; D002942:Circular Dichroism; D004722:Endoribonucleases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics*; D029968:Escherichia coli Proteins; D019793:Fluorescein / Q000378:metabolism; D005453:Fluorescence; D007700:Kinetics; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D009691:Nucleic Acid Denaturation; D012322:RNA Precursors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012329:RNA, Bacterial / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D016337:RNA, Catalytic / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D012353:RNA, Transfer, Gly / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D043262:Ribonuclease P; D013696:Temperature; D013816:Thermodynamics
D029968:Escherichia coli Proteins; D012322:RNA Precursors; D012329:RNA, Bacterial; D016337:RNA, Catalytic; D012353:RNA, Transfer, Gly; D004722:Endoribonucleases; D043262:Ribonuclease P; C470865:ribonuclease P, E coli; D019793:Fluorescein