Selection of small molecules by the Tetrahymena catalytic center.
The catalytic center in group I RNAs contains a selective binding site that accommodates both guanosine and L-arginine. In order to understand the specificity of the RNA for small molecules, we analyzed 6 RNAs that vary in this region. Specificity for nucleotides resides substantially in G264 rather than its paired nucleotide C311, and is expressed substantially in Km, with comparatively little variation in kcat. kcat is not notably perturbed even for RNAs with mispairs in the active-site helix. For 5 of 6 sequences, effects of RNA substitutions on arginine binding and GTP reactivity are proportional, confirming that arginine contacts a subset of the groups occupied by G. As a result of particular mutations, reaction with GTP is decreased, and reaction with the natural nucleotides UTP and ATP is enhanced. Molecular modeling of these effects suggests that exceptionally flexible placement of reactants may be an essential quality of RNA-catalyzed splicing. The specificity of the intron can be rationalized by a type of binding model not previously considered, in which the G/arginine site includes adjacent nucleotides (an 'axial' site), rather than a single nucleotide, G264.
Yarus
M
M
Department of Molecular, Cellular and Development Biology, University of Colorado, Boulder 80309-0347.
Illangesekare
M
M
Christian
E
E
D000596:Amino Acids / Q000737:chemistry; D000818:Animals; D001120:Arginine / Q000737:chemistry; D001483:Base Sequence; D002384:Catalysis; D002853:Chromatography, Liquid; D006160:Guanosine Triphosphate / Q000737:chemistry; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D012326:RNA Splicing; D016053:RNA, Protozoan / Q000737:chemistry*; D013379:Substrate Specificity; D013768:Tetrahymena / Q000378:metabolism*
D000596:Amino Acids; D016053:RNA, Protozoan; D006160:Guanosine Triphosphate; D001120:Arginine