Guanosine binds to the Tetrahymena ribozyme in more than one step, and its 2'-OH and the nonbridging pro-Sp phosphoryl oxygen at the cleavage site are required for productive docking.
The dynamics of binding of various guanosine, or G, substrates to the Tetrahymena thermophila L-21 ScaI ribozyme have been investigated by fluorescence-detected stopped-flow experiments. Upon rapid mixing of various G substrates with a preformed complex of the ribozyme and the fluorescent 5' splice site analogue CCUCUepsilonA, fluorescence transients that provide rates for binding of G substrates and the rate-limiting step for transesterification are observed. The measured apparent bimolecular rate constant for binding of pG is 10(3) M-1 s-1, much slower than expected for diffusion. pG appears to bind to the preformed complex of the ribozyme and CCUCUepsilonA in at least two steps, a bimolecular step followed by at least one conformational change. This two-step binding of pG, involving a rapid pre-equilibrium, leads to the slow apparent rate constant for binding of pG. Furthermore, the 2'-OH of pG and of the 3' terminal G of the G substrate GUCG and the nonbridging pro-Sp phosphoryl oxygen atom at the site of phosphoryl transfer on CCUCUepsilonA appear to mediate formation of a properly conformed docked ternary complex of the G substrate, 5' splice site, and ribozyme which may represent an intermediate required for initiation of transesterification. It is possible that the 2'-OH of pG and this nonbridging pro-Sp phosphoryl oxygen interact, directly or indirectly, with one another.
Profenno
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Department of Chemistry, Box 270216, University of Rochester, Rochester, New York 14627-0216, USA.
Kierzek
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Testa
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Turner
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D000818:Animals; D001665:Binding Sites; D006151:Guanosine / Q000378:metabolism*; D009690:Nucleic Acid Conformation; D010100:Oxygen / Q000378:metabolism; D010766:Phosphorylation; D016337:RNA, Catalytic / Q000378:metabolism*; D016808:Tetrahymena thermophila / Q000235:genetics / Q000378:metabolism*
D016337:RNA, Catalytic; D006151:Guanosine; D010100:Oxygen