Poster abstracts

Poster number 31 submitted by Hajeong Kim

Biochemical and thermodynamic characterization of guanosine substrate-binding of a group I Intron from bacteriophage Twort

Hajeong Kim (Department of Biochemistry, Purdue University), Josh Sokoloski (Department of Chemistry, Penn State University), Philip C. Bevilacqua (Department of Chemistry, Penn State University), Barbara L. Golden (Department of Biochemistry, Purdue University)

Abstract:
The group I intron from the bacteriophage Twort binds guanosine (G) very tightly, much tighter than the Anabaena and Tetrahymena group I introns. This is surprising because group I intron active sites are very highly conserved. To explore why this intron might bind G so tightly, we first examined the possibility that these differences are originated from a little sequence variation in G-binding site. In a Twort mutant ribozyme that has the sequences of the top layer of G-binding site of the Tetrahymena ribozyme, guanosine dissociation constant increases 4-fold. This is consistent with the finding that in the Tetrahymena ribozyme, there is less stacking between G-substrate and the active site residues than in other ribozymes. As stacking is one of the major forces to stabilize nucleic acid structure, this partly explains our inquiry. To further examine the origin of the differences in G binding, we characterized the thermodynamics of G binding by isothermal titration calorimetry and kinetic experiments. Surprisingly, the Twort ribozyme behaves differently than other group I introns. The Tetrahymena and Anabaena ribozymes bind G in an entropically favored process where î H = ~0. In contrast, G binding by the Twort intron is enthalpically favored. To explain these unexpected differences, we hypothesize that the unbound conformation of G-binding site of the Twort ribozyme is more native-like while in the other group I introns, the site is nearly collapsed. This may be due at least in part to the presence of P7.1-7.2 subdomain that may stabilize the conformation of the active site.
Neurospora mt tyrosyl-tRNA synthetase protein (CYT-18) recognizes and stabilizes the core structure of group I introns. Several lines of evidence suggest that CYT-18 stabilizes the conformation of the G-binding site of group I introns. To examine this possibility, we determined Mg2+ concentration dependence of G-binding constant (KMG). While KMG in the absence of CYT-18 was greatly increased with decreasing Mg2+concentration, in the CYT-18-assisted reaction, it was nearly constant. This suggests that CYT-18 stabilizes the three-dimensional structure of G-binding site at low Mg2+concentrations. The number of Mg2+ions required for catalysis was estimated by studying the Mg2+concentration dependence of kcat. The number of Mg2+ions binding in the absence of CYT-18 is much higher than that in the presence of CYT-18. This suggests that influence of structural Mg2+ions be at least lowered in presence of Cyt-18, but that the protein cannot compensate for catalytic Mg2+ ions. This system may allow us to probe the roles of the catalytic Mg2+ions.

Keywords: group I Intron, thermodynamics, guanosine binding