6384 zyxwvutsrqpon Biochemistry zyxwvu 1988, 27, 6384-6392 Kinetics for Reaction of a Circularized Intervening Sequence with CU, UCU, Temperature and on Oligomer and MgZf Concentrationst CUCU, and CUCUCU: Mechanistic Implications from the Dependence on Naoki Sugimoto,$ Ryszard Kierzek,§ and Douglas H. Turner*,! Department zyxwvutsrq of Chemistry, University of Rochester, Rochester, New York 14627, and Institute of Bioorganic Chemistry, Polish Academy of Sciences, 60- 704 Poznan, Noskowskiego 12114, Poland Received September 29, 1987; Revised Manuscript Received March 24, 1988 zyxwvutsrqponmlkjihgfedcbaZYXW ABSTRACT: 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 oligonucleotides 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 zyxwv OC in 1 and 10 mM Mg2+ are zyxw 5 X lo3 and 2.5 X lo4 M-l, respectively. These are roughly 4 orders of magnitude larger than expected for simple Wat- son-Crick base pairing. The binding constants derived for UCU, CUCU, and CUCUCU at 30 "C in 10 mM Mg2+ are 1.2 X los, 4 X lo5, and >lo7 M-l, respectively. The free energy increments for binding of UCU and CUCU relative to C U 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. The binding constant derived for Mg2+ in the presence of CU is 510 M-' at 30 "C and is almost temperature independent. The temperature dependence is similar to that reported for other Mg2+.RNA complexes. The binding constant, however, is smaller than most binding constants for Mg2+ to RNA, except for complexes involving inner-sphere complexation. The results suggest reverse cyclization of C IVS may involve binding of a weakly held Mg2+ion. At 30 OC, rate constants for the reaction step involving transesterification with CU average roughly 0.05 and 0.4 h-l, respectively, for cyclization and reverse cyclization. The rate for reverse cyclization is essentially independent of oligomer length. This indicates the rate of transesterification is not limited by the rate for disruption of the pairing between substrate and C IVS. The activation energy and entropy for reverse cyclization with CU are unusually large, 50 kcal/mol and 86 eu, respectively. The large, positive zyxwvu AS* indicates the transition state for the rate-determining step is less ordered than the Mg2+.C IVSCU intermediate. This raises the possibility that a partial unfolding of the C IVS structure occurs during reaction. Thus, the "transesterification step" probably involves additional intermediates. The large activation energy indicates it should be possible to trap and study the Mg2+-C IVS-CU intermediate. It is known that RNA is able to catalyze the making and breaking of covalent bonds (Kruger et al., 1982; Zaug zyxwvuts & Cech, 1986; Guerrier-Takada & Altman, 1984). The detailed mechanisms of these reactions are the subject of continued investigation. The first RNA known to facilitate cleavage and ligation reactions in RNA is the intervening sequence (IVS) from the rRNA precursor of Tetrahymena thermophila (Kruger et al., 1982; Cech & Bass, 1986). This RNA me- diates a self-excision coupled to an exon splicing reaction and subsequently undergoes a self-circularization reaction (Zaug et al., 1983). The circular product (C IVS) can be linearized by addition of oligoribonucleotides, and this new product is itself able to undergo circularization in a reversible manner (Sullivan & Cech, 1985). These last reactions provide a convenient model system for studying the mechanism of IVS reactions. Since all the reactions are transesterifications, it is likely the mechanisms are similar. 'This work was supported by National Institutes of Health Grant GM *Address correspondence to this author. *University of Rochester. $ Polish Academy of Sciences. zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA 22939. In this paper, we report the kinetics in the minutes to hours time range for the linearization of C IVS with the oligoribo- nucleotides CU, UCU, CUCU, and CUCUCU. The kinetics as a function of oligomer and Mg2+ concentration and of temperature suggest the mechanism involves separate binding steps for both oligomer and Mg2+. The binding constants deduced for the oligomers are anomalously large. The anomalous component of the binding is apparently restricted to two nucleotides, however. The binding constant deduced for Mg2+ is small, but consistent with formation of an in- ner-sphere complex. The activation energy and entropy for reverse cyclization are unusually large and raise the possibility that a conformational change is required to form the transition state. MATERIALS AND METHODS Nucleic Acids. CU was obtained from Sigma, and purity was confirmed by high-performance liquid chromatography. UCU, CUCU, and CUCUCU were synthesized on solid support with a phosphoramidite method (Kierzek et al., 1986) and purified by high-performance liquid chromatography (Ikuta et al., 1984). Concentrations were determined optically with extinction coefficients of 1.67 X lo4, 2.66 X IO4, 3.33 X 0006-2960/88/0427-6384$01.50/0 zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA 0 1988 American Chemical Society