Guanosine Binds to the Tetrahymena Ribozyme in More than One Step, and Its 2′-OH and the Nonbridging pro-S p Phosphoryl Oxygen at the Cleavage Site Are Required for Productive Docking ² Louis A. Profenno, ‡ Ryszard Kierzek, § Stephen M. Testa, ‡ and Douglas H. Turner* ,‡ Department of Chemistry, Box 270216, UniVersity of Rochester, Rochester, New York 14627-0216, and Institute of Bioorganic Chemistry, Polish Academy of Sciences, 60-704 Poznan, Noskowskiego 12/14, Poland ReceiVed April 16, 1997; ReVised Manuscript ReceiVed August 11, 1997 X ABSTRACT: 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 CCUCUǫA, 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 CCUCUǫA 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-S p phosphoryl oxygen atom at the site of phosphoryl transfer on CCUCUǫA 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-S p phosphoryl oxygen interact, directly or indirectly, with one another. Little is known about the dynamics of natural RNAs in either folding or function. The kinetics of RNA folding have been studied for tRNA (Crothers et al., 1974; Hilbers et al., 1976) and for the Tetrahymena thermophila L-21 ScaI ribozyme (Zarrinker & Williamson, 1994; Banerjee & Turner, 1995; Sclavi et al., 1997). RNA dynamics related to function have been studied for a spliced leader sequence (Le Cuyer & Crothers, 1994) and for the Tetrahymena L-21 ScaI ribozyme (Bevilacqua et al., 1992, 1993, 1994; Li et al., 1995; Li & Turner, 1997; Turner et al., 1996). Much remains to be discovered, however, about the factors that affect RNA dynamics. The T. thermophila L-21 ScaI ribozyme, R, is a version of the T. thermophila LSU intron shortened by 21 and 5 nucleotides at the 5′ and 3′ ends, respectively. It catalyzes a transesterification reaction between an exogenously added analogue of the 5′ splice site and the guanosine, or G, substrate (Inoue & Kay, 1987; Zaug et al., 1988). A2′-H substitution on G and a sulfur substitution for the nonbridg- ing, pro-S p phosphoryl oxygen atom at the site of transes- terification inhibit the transesterification reaction catalyzed by this RNA (Bass & Cech, 1986; Rajagopal et al., 1989; Piccirilli et al., 1993). A mechanism for explaining inhibition by either substitution has not been elucidated, however. An understanding of how these substitutions affect the dynamics of individual steps required for catalysis will offer insights into the catalytic mechanism of RNA. For either substitution, inhibition could be due to an effect on substrate binding and positioning and/or to an effect on the phosphoryl transfer step. Fluorescent probes of substrate binding and phosphoryl transfer offer a means of following individual steps in the catalytic pathway (Bevilacqua et al., 1992, 1994; Li et al., 1995; Turner et al., 1996) and, therefore, of potentially elucidating how these modifications on substrates affect catalysis. Previous studies have used a pyrene probe at the 5′ end of an oligonucleotide substrate to follow docking of a helix into the catalytic site. As with most fluorescent probes of RNA (Tuschl et al., 1994; Walter & Burke, 1997), the pyrene probe is large, consisting of 38 atoms. Here, we report the application of a much smaller probe, 1,N 6 -ethenoadenosine, ǫA (Figure 1) (Tolman et al., 1974; Spencer et al., 1974), to the study of steps involved in the binding of various G substrates to the T. thermophila L-21 ScaI ribozyme. The ǫA adds only four new atoms to a5′ splice site analogue and is at the 3′ rather than at the 5′ end of the substrate. A kinetic analysis of interactions between various G substrates and a preformed complex of the 5′ splice site analogue CCUCUǫA, and its phospho- rothioate derivatives, with the L-21 ScaI ribozyme has been undertaken. Changes in the environment of ǫA lead to changes in ǫA fluorescence (Tolman et al., 1974; Spencer et al., 1974; Kubota et al., 1983). Herein, fluorescence transients are shown to result upon binding of the G substrate to the complex of the ribozyme and CCUCUǫA. By analyzing the fluorescence transients induced by different G substrates, we infer that the G substrate binds in at least two steps to the complex of the ribozyme and CCUCUǫA and that the 2′-OH of the G substrate and the nonbridging, pro-S p phosphoryl oxygen at the site of phosphoryl transfer ² This work was supported by NIH Grant GM22939. * To whom correspondence should be addressed. ‡ University of Rochester. § Polish Academy of Sciences. X Abstract published in AdVance ACS Abstracts, September 15, 1997. 12477 Biochemistry 1997, 36, 12477-12485 S0006-2960(97)00889-1 CCC: $14.00 © 1997 American Chemical Society