title: Tertiary interactions with the internal guide sequence mediate docking of the P1 helix into the catalytic core of the Tetrahymena ribozyme.
abstract: The L-21 ScaI ribozyme catalyzes sequence-specific cleavage of an oligonucleotide substrate. Cleavage is preceded by base pairing of the substrate to the internal guide sequence (IGS) at the 5' end of the ribozyme to form a short RNA duplex (P1). Tertiary interactions between P1 and the catalytic core dock P1 into the active site of the ribozyme. These include interactions between the catalytic core and 2'-hydroxyls of the substrate at nucleotide positions -3u and perhaps -2c. In this study, 2'-hydroxyls of the IGS strand that contribute to P1 recognition by the ribozyme are identified. IGS 2'-hydroxyls (nucleotide positions 22-27) were individually modified to either 2'-deoxy or 2'-methoxynucleotides within full-length semisynthetic L-21 ScaI ribozymes generated using T4 DNA ligase. Thermodynamic and kinetic characterization of the resulting IGS variant ribozymes justify the following conclusions: (i) 2'-Hydroxyls at nucleotide positions G22 and G25 play a critical energetic role in docking P1 into the catalytic core, contributing 2.6 and 2.1 kcal.mol-1, respectively. (ii) The loss of binding energy is manifest primarily as an increase in the rate of dissociation. Because turnover for the wild-type ribozyme is limited by product dissociation, G22 and G25 deoxy variants display up to a 20-fold increase in the multiple-turnover rate at saturating substrate. (iii) IGS tertiary interactions are energetically coupled with the tertiary interactions made to the substrate, consistent with P1 becoming undocked from its binding site in J8/7 upon substitution of either the G22 or G25 2'-hydroxyl. (iv) The G22 deoxy variant loses energetic coupling between guanosine and substrate binding, suggesting that in this variant the P1 helix is also undocked from its binding site in J4/5, the proposed site of guanosine and substrate interaction. Therefore, in combinations with previous studies four P1 2'-hydroxyls are implicated as important for docking. The contributions of the 2'-hydroxyl tertiary interactions are not equivalent and follow the hierarchical order G22 > G25 >> -3u > -2c. Because the G22 2'-hydroxyl appears to mediate P1 docking into both J8/7 and J4/5, it may serve as the molecular linchpin for the recognition of P1 by the catalytic core.
authors.affiliation: Howard Hughes Medical Institute, Department of Chemistry and Biochemistry, University of Colorado, Boulder 80309-0215.
authors.forename: S A
authors.initials: SA
authors.lastname: Strobel
authors.forename: T R
authors.initials: TR
authors.lastname: Cech
mesh_terms: D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D011088:DNA Ligases / Q000378:metabolism; D004355:Drug Stability; D006860:Hydrogen Bonding; D006900:Hydroxylation; D007438:Introns; D007700:Kinetics; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D012313:RNA / Q000737:chemistry* / Q000378:metabolism*; D016337:RNA, Catalytic / Q000737:chemistry* / Q000378:metabolism*; D013329:Structure-Activity Relationship; D016808:Tetrahymena thermophila / Q000235:genetics*; D013816:Thermodynamics
chemical_list: D016337:RNA, Catalytic; D012313:RNA; D011088:DNA Ligases