Effects of substrate structure on the kinetics of circle opening reactions of the self-splicing intervening sequence from Tetrahymena thermophila: evidence for substrate and Mg2+ binding interactions.
The self-splicing intervening sequence from the precursor rRNA of Tetrahymena thermophila cyclizes to form a covalently closed circle. This circle can be reopened by reaction with oligonucleotides or water. The kinetics of circle opening as a function of substrate and Mg2+ concentrations have been measured for dCrU, rCdU, dCdT, and H2O addition. Comparisons with previous results for rCrU suggest: (1) the 2' OH of the 5' sugar of a dinucleoside phosphate is involved in substrate binding, and (2) the 2' OH of the 3' sugar of a dimer substrate is involved in Mg2+ binding. Evidently, the binding site for a required Mg2+ ion is dependent on both the ribozyme and the dimer substrate. The apparent activation energy and entropy for circle opening by hydrolysis are 31 kcal/mol and 50 eu, respectively. The large, positive activation entropy suggests a partial unfolding of the ribozyme is required for reaction.
Sugimoto
N
N
Department of Chemistry, University of Rochester, NY 14627.
Tomka
M
M
Kierzek
R
R
Bevilacqua
P C
PC
Turner
D H
DH
D000818:Animals; D015226:Dinucleoside Phosphates / Q000378:metabolism*; D007438:Introns; D007700:Kinetics; D008274:Magnesium / Q000378:metabolism*; D008433:Mathematics; D008962:Models, Theoretical; D012322:RNA Precursors / Q000235:genetics* / Q000378:metabolism; D012326:RNA Splicing; D016337:RNA, Catalytic; D012335:RNA, Ribosomal / Q000235:genetics* / Q000378:metabolism; D013768:Tetrahymena / Q000235:genetics*; D013816:Thermodynamics
D015226:Dinucleoside Phosphates; D012322:RNA Precursors; D016337:RNA, Catalytic; D012335:RNA, Ribosomal; D008274:Magnesium