MONITORING OF THE COOPERATIVE UNFOLDING OF THE STATE-UNIVERSITY-OF-NEW-YORK GROUP-I INTRON OF BACTERIOPHAGE-T4 - THE ACTIVE FORM OF THE STATE-UNIVERSITY-OF-NEW-YORK RIBOZYME IS STABILIZED BY MULTIPLE INTERACTIONS WITH 3' TERMINAL INTRON COMPONENTS

We have studied the mechanism by which the 3′ terminal domain of the sunY intron of bacteriophage T4 activates the group I ribozyme core of this intron, from which it is separated by some 800 nucleotides. As shown by monitoring either UV absorbance or self-splicing reaction kinetics as a function of temperature, intron transcripts undergo highly cooperative unfolding/inactivation upon heating: the two methods yield similar estimates of the thermodynamic parameters associated with this process. Such cooperativity makes it possible in turn to assess the energetic contribution of specific interactions to the overall structure, by comparing the sensitivity to heat inactivation of molecules carrying various nucleotide substitutions. By combining this approach with chemical modification, we have probed several proven or putative interactions between the core and 3′ terminal domain of the intron and conclude that the role of the 3′ terminal domain is to stabilize the active form of the ribozyme. Interestingly, the P9.0 interaction, which brings 3′ terminal nucleotides next to the core site that binds the guanosine cofactor of the self-splicing reaction, is now shown to be composed in fact of two distinct pairings. An isolated base-pair (P9.0a), involving a residue located only six nucleotides upstream of the 3′ splice site, participates in the stabilization of the ribozyme and appears to persist during the second stage of self-splicing (exon ligation). In contrast, formation of the previously demonstrated P9.0b pairing, which involves the two penultimate intron nucleotides, contributes no additional stability and results in no detectable rearrangement of the core structure. Implications for the concept of a static ribozyme are discussed in the light of a slightly revised three-dimensional model of the sunY intron. © 1993 Academic Press Limited.