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Mutations in the Tetrahymena ribozyme internal guide sequence: effects on docking of the P1 helix into the catalytic core and correlation with catalytic activity.
Biochemistry. 1996 Sep 03; 35(35):11493-502.B

Abstract

Binding of substrate by the ribozyme derived from the self-splicing intron of Tetrahymena thermophila involves at least two steps. In the first step, base pairing between the ribozyme internal guide sequence (IGS) and the substrate forms a helical duplex (P1). Through specific tertiary interactions between P1 and the ribozyme core, P1 is then docked into the ribozyme active site. We have investigated the effects of compensatory mutations in positions 2-6 of the P1 helix on docking of P1 into the ribozyme core. Equilibrium binding of matching oligonucleotides by catalytically active IGS mutant ribozymes was evaluated by gel-shift analysis. While the strength of base pairing changed with base composition as expected, the strength of tertiary interactions between P1 and the ribozyme core was not affected by the P1 mutations. These results support a model in which efficient docking of P1 is determined by P1 structure and the presence of a conserved G-U pair. Determination of the rate of dissociation of matching oligonucleotides from each ribozyme revealed that mutations in the IGS change the tightness of binding by increasing or decreasing the dissociation rate. Surprisingly, dissociation rates determined in this fashion were 20-900-fold less than the values of the multiple-turnover rate constant for these ribozymes, initially suggesting that turnover did not require product dissociation. A more detailed analysis for the wild-type ribozyme defined two distinct product dissociation rates. The slower rate equaled that determined under the conditions used for the equilibrium binding studies. The weighted average of the two dissociation rates equaled the multiple-turnover rate constant. These results are explained by a model in which ribozyme preparations consist of two ribozyme conformers: one with tight docking of P1 and another with weaker docking of P1.

Authors+Show Affiliations

Howard Hughes Medical Institute, Department of Chemistry and Biochemistry, University of Colorado, Boulder 80309, USA.No affiliation info available

Pub Type(s)

Journal Article
Research Support, Non-U.S. Gov't
Research Support, U.S. Gov't, P.H.S.

Language

eng

PubMed ID

8784205

Citation

Campbell, T B., and T R. Cech. "Mutations in the Tetrahymena Ribozyme Internal Guide Sequence: Effects On Docking of the P1 Helix Into the Catalytic Core and Correlation With Catalytic Activity." Biochemistry, vol. 35, no. 35, 1996, pp. 11493-502.
Campbell TB, Cech TR. Mutations in the Tetrahymena ribozyme internal guide sequence: effects on docking of the P1 helix into the catalytic core and correlation with catalytic activity. Biochemistry. 1996;35(35):11493-502.
Campbell, T. B., & Cech, T. R. (1996). Mutations in the Tetrahymena ribozyme internal guide sequence: effects on docking of the P1 helix into the catalytic core and correlation with catalytic activity. Biochemistry, 35(35), 11493-502.
Campbell TB, Cech TR. Mutations in the Tetrahymena Ribozyme Internal Guide Sequence: Effects On Docking of the P1 Helix Into the Catalytic Core and Correlation With Catalytic Activity. Biochemistry. 1996 Sep 3;35(35):11493-502. PubMed PMID: 8784205.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Mutations in the Tetrahymena ribozyme internal guide sequence: effects on docking of the P1 helix into the catalytic core and correlation with catalytic activity. AU - Campbell,T B, AU - Cech,T R, PY - 1996/9/3/pubmed PY - 1996/9/3/medline PY - 1996/9/3/entrez SP - 11493 EP - 502 JF - Biochemistry JO - Biochemistry VL - 35 IS - 35 N2 - Binding of substrate by the ribozyme derived from the self-splicing intron of Tetrahymena thermophila involves at least two steps. In the first step, base pairing between the ribozyme internal guide sequence (IGS) and the substrate forms a helical duplex (P1). Through specific tertiary interactions between P1 and the ribozyme core, P1 is then docked into the ribozyme active site. We have investigated the effects of compensatory mutations in positions 2-6 of the P1 helix on docking of P1 into the ribozyme core. Equilibrium binding of matching oligonucleotides by catalytically active IGS mutant ribozymes was evaluated by gel-shift analysis. While the strength of base pairing changed with base composition as expected, the strength of tertiary interactions between P1 and the ribozyme core was not affected by the P1 mutations. These results support a model in which efficient docking of P1 is determined by P1 structure and the presence of a conserved G-U pair. Determination of the rate of dissociation of matching oligonucleotides from each ribozyme revealed that mutations in the IGS change the tightness of binding by increasing or decreasing the dissociation rate. Surprisingly, dissociation rates determined in this fashion were 20-900-fold less than the values of the multiple-turnover rate constant for these ribozymes, initially suggesting that turnover did not require product dissociation. A more detailed analysis for the wild-type ribozyme defined two distinct product dissociation rates. The slower rate equaled that determined under the conditions used for the equilibrium binding studies. The weighted average of the two dissociation rates equaled the multiple-turnover rate constant. These results are explained by a model in which ribozyme preparations consist of two ribozyme conformers: one with tight docking of P1 and another with weaker docking of P1. SN - 0006-2960 UR - https://www.unboundmedicine.com/medline/citation/8784205/Mutations_in_the_Tetrahymena_ribozyme_internal_guide_sequence:_effects_on_docking_of_the_P1_helix_into_the_catalytic_core_and_correlation_with_catalytic_activity_ L2 - https://doi.org/10.1021/bi960510z DB - PRIME DP - Unbound Medicine ER -