Cooperative and anticooperative binding to a ribozyme.

Abstract

The effects of guanosine 5'-monophosphate and 2'-deoxyguanosine 5'-monophosphate on the thermodynamics and kinetics ofpyrene-labeled 5' exon mimic (pyCUCU) binding to the catalytic RNA (ribozyme) from Tetrahymena thermophila have been determined by fluorescence titration and kinetics experiments at 15°C. pyCUCU binding to L-21 Sca I-truncated ribozyme is weaker by a factor of 5 in the presence of saturating guanosine 5'-monophosphate, whereas it is 4-fold stronger in the presence of saturating 2'-deoxyguanosine 5'-monophosphate. Results from kinetics experiments suggest that anticooperative effects in the presence of guanosine 5'-monophosphate arise primarily from slower formation of tertiary contacts between the catalytic core of the ribozyme and the P1 duplex formed by pyCUCU and GGAGGG of the ribozyme. Conversely, cooperative effects in the presence of 2'-deoxyguanosine 5'-monophosphate arise primarily from slower disruption oftertiary contacts between the catalytic core of the ribozyme and the P1 duplex. Additional experiments suggest that these cooperative and anticooperative effects are not a function ofthe pyrene label, are not caused by a salt effect, and are not specific to one renaturation procedure for the ribozyme.Regulation of enzyme activity is often mediated by cooperative and anticooperative interactions among substrates. Coupling of substrate binding has been the subject of many theoretical investigations (1-4). Substrate interactions allow enzymes to respond to changes in their environment (5-8). Indeed, the activity of several enzymes is regulated by the binding of a single nucleotide cofactor (9-12). The L-21 Sca I-truncated catalytic RNA (ribozyme), designated "L-21 Sca I," derived from the self-splicing LSU intron of Tetrahymena thermophila has two substrate-binding sites: one for the nucleotide guanosine and its 5'-phosphate derivatives and one for 5'-splice-site mimics, including CUCU (13,33), opening the possibility for heterotropic substrate interactions. In addition, the G-binding site can also accommodate dG, a competitive inhibitor of G (14).Recent experiments on mutants of L-21 Sca I (15) and stopped-flow experiments with fluorescent pyrene derivatives of 5' exon mimics (16,17) established that 5'-exon mimics bind to L-21 Sca I in two steps. The first step is base pairing to GGAGGG (the internal guide sequence) to form the P1 pairing (18,19), and the second step is formation oftertiary contacts between P1 and the catalytic core of the ribozyme (Fig. 1). At least some of these tertiary contacts involve specific 2'-OH groups of the 5'-exon mimic at the underlined positions: CUCU (20, 21).We report here that the nucleotide cofactor guanosine 5'-monophosphate (pG) causes pyrene-labeled CUCU (py-CUCU) binding to L-21 Sca I to be weaker by a factor of 5 (+0.9 kcal/mol at 15°C), whereas the inhibitor 2'-deoxy-The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C...

Guanosine binding to the Tetrahymena ribozyme: thermodynamic coupling with oligonucleotide binding.

“…3, which show binding of G being destabilized in the presence of saturating CCCUCU. This destabilization is consistent with data in the accompanying paper (19).…”

The P5abc Peripheral Element Facilitates Preorganization of the Tetrahymena Group I Ribozyme for Catalysis

“…This cooperativity is absent for E wt (69), consistent with greater preorganization of the wild-type active site.…”

Group I Ribozymes: Substrate Recognition, Catalytic Strategies, and Comparative Mechanistic Analysis

“…3) then docks into the active site cleft (Bevilacqua et al 1992;Herschlag 1992) via multiple tertiary interactions with the backbone and the G -U pair at the cleavage site (see Strobel and Cech 1995 and references therein). Guanosine binding is unordered with respect to binding of S (Herschlag and Cech 1990a), although there is modest (-1 kcal/mol) energetic coupling between the two (Bevilacqua et al 1993;McConnell et al 1993). Once the ternary complex (EaG-S) is formed, transesterification occurs very rapidly (-300min-1 at 50 "C or 80min-' at 30°C; Herschlag andCech 1990a: McConnell et al 1993;Herschlag and Khosla 1994).…”