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

被引:58
作者
Engelhardt, MA
Doherty, EA
Knitt, DS
Doudna, JA
Herschlag, D [1 ]
机构
[1] Stanford Univ, Dept Biochem, Stanford, CA 94305 USA
[2] Yale Univ, Dept Mol Biophys & Biochem, New Haven, CT 06520 USA
[3] Yale Univ, Howard Hughes Med Inst, New Haven, CT 06520 USA
关键词
D O I
10.1021/bi992313g
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
Phylogenetic comparisons and site-directed mutagenesis indicate that group I introns are composed of a catalytic core that is universally conserved and peripheral elements that are conserved only within intron subclasses. Despite this low overall conservation, peripheral elements are essential for efficient splicing of their parent introns. We have undertaken an in-depth structure-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 cleavage 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 global tertiary structure at Mg2+ concentrations greater than 20 mM [Doherty, E. A., et al. (1999) Biochemistry 38, 2982-90]. However, free E-Delta P5abc is 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 positions in the conserved core that cluster around the substrate binding sites. Kinetic and thermodynamic dissection of individual reaction steps identified 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 greater than or equal to 350-fold weaker docking of the oligonucleotide substrate into its tertiary interactions with the ribozyme core. These defects in binding of the substrates account for essentially all of the 10(4)-fold decrease 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 indirect interactions, thereby preferentially stabilizing the active ribozyme structure relative to alternative less active states. This is consistent with the view that peripheral elements engage in a network of mutually reinforcing interactions that together ensure cooperative folding of the ribozyme to its active structure.
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页码:2639 / 2651
页数:13
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