Kinetics for reaction of a circularized intervening sequence with CU, UCU, CUCU, and CUCUCU: mechanistic implications from the dependence on temperature and on oligomer and Mg2+ concentrations.
The self-splicing intervening sequence from the rRNA precursor in Tetrahymena thermophila produces a covalently closed, circularized form (C IVS). Reaction rates for reverse cyclization (linearization) of C IVS by the covalent addition of the oligoncleotides CU, UCU, CUCU, and CUCUCU have been measured. The dependence of the observed rates on oligomer and Mg2+ concentrations indicates the presence of intermediates that are generated by separate binding steps for both oligomer and Mg2+. Linearization of C IVS by OH- hydrolysis is suppressed in the presence of oligomer, suggesting oligomer binds near the active site. The binding constants derived for CU at 30 degrees C in 1 and 10 mM Mg2+ are 5 X 10(3) and 2.5 X 10(4) M-1, respectively. These are roughly 4 orders of magnitude larger than expected for simple Watson-Crick base pairing. The binding constants derived for UCU, CUCU, and CUCUCU at 30 degrees C in 10 mM Mg2+ are 1.2 X 10(5), 4 X 10(5), and greater than 10(7) M-1, respectively. The free energy increments for binding of UCU and CUCU relative to CU are similar to those expected from a nearest-neighbor model for addition of base pairs. This indicates the factors responsible for the unusually strong binding of CU to C IVS are restricted to two nucleotides.(ABSTRACT TRUNCATED AT 250 WORDS)
Sugimoto
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N
Department of Chemistry, University of Rochester, New York 14627.
Kierzek
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R
Turner
D H
DH
D000818:Animals; D001483:Base Sequence; D007438:Introns; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology*; D008433:Mathematics; D008962:Models, Theoretical; D009691:Nucleic Acid Denaturation; D009843:Oligoribonucleotides; D012313:RNA / Q000235:genetics*; D012322:RNA Precursors / Q000235:genetics*; D012326:RNA Splicing / Q000187:drug effects; D000079962:RNA, Circular; D012335:RNA, Ribosomal / Q000235:genetics*; D013768:Tetrahymena / Q000235:genetics*; D013816:Thermodynamics
D009843:Oligoribonucleotides; D012322:RNA Precursors; D000079962:RNA, Circular; D012335:RNA, Ribosomal; D012313:RNA; D008274:Magnesium