The P5abc peripheral element facilitates preorganization of the Tetrahymena group I ribozyme for catalysis

Phylogenetic comparisons and site-directed mutagenesis indicate that group I introns are composed of a catalytic core that is universally conserved an d peripheral elements that are conserved only within intron subclasses. Des pite this low overall conservation, peripheral elements are essential for e fficient splicing of their parent introns. We have undertaken an in-depth s tructure-function analysis to investigate the role of one of these elements , P5abc, using the well-characterized ribozyme derived from the Tetrahymena group I intron. structural comparisons using solution-based free radical c leavage revealed that a ribozyme lacking P5abc (E-Delta P5abc) and E-Delta P5abc With P5abc added in trans (E-Delta P5abc.P5abc) adopt a similar globa l tertiary structure at Mg2+ concentrations greater than 20 mM [Doherty, E. A., et al. (1999) Biochemistry 38, 2982-90]. However, free E-Delta P5abc i s greatly compromised in overall oligonucleotide cleavage activity, even at Mg2+ concentrations as high as 100 mM. Further characterization of E-Delta P5abc, DMS modification revealed local structural differences at several p ositions in the conserved core that cluster around the substrate binding si tes. Kinetic and thermodynamic dissection of individual reaction steps iden tified defects in binding of both substrates to E-Delta P5abc, With greater than or equal to 25-fold weaker binding of a guanosine nucleophile and gre ater than or equal to 350-fold weaker docking of the oligonucleotide substr ate into its tertiary interactions with the ribozyme core. These defects in binding of the substrates account for essentially all of the 10(4)-fold de crease in overall activity of the deletion mutant. Together, the structural and functional observations suggest that the P5abc peripheral element not only provides stability but also positions active site residues through ind irect interactions, thereby preferentially stabilizing the active ribozyme structure relative to alternative less active states. This is consistent wi th the view that peripheral elements engage in a network of mutually reinfo rcing interactions that together ensure cooperative folding of the ribozyme to its active structure.