KINETICS AND THERMODYNAMICS OF TRIPLE-HELIX FORMATION - EFFECTS OF IONIC-STRENGTH AND MISMATCHES

被引:255
作者
ROUGEE, M [1 ]
FAUCON, B [1 ]
MERGNY, JL [1 ]
BARCELO, F [1 ]
GIOVANNANGELI, C [1 ]
GARESTIER, T [1 ]
HELENE, C [1 ]
机构
[1] UNIV ILLES BALEARS, DEPT BIOL & CIENCIES SALUT, PALMA DE MALLORCA, SPAIN
关键词
D O I
10.1021/bi00153a021
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
Thermodynamic and kinetic parameters for the triplex-forming reactions between a homopurine-homopyrimidine 22-base-pair duplex (sequence of the purine strand: 5'd[AAAGGAGGAGAAGAAGAAAAAA]3') and the four 22-dN third strands (22 dN: 5'd[TTTCCTCCTCTNCTTCTTTTTT]3', where N = A, C, T, or G) were determined from thermal denaturation and renaturation UV absorbance profiles. Cooling and heating curves were not superimposable and thus allowed us to determine the rate constants of association (k(on)) and dissociation (k(off)) as a function of temperature, assuming a two-state model analogous to that developed for duplex-forming reactions. Experiments were performed in 10 mM cacodylate buffer (pH 6.8) in the presence of NaCl concentrations ranging from 20 to 300 mM. Within experimental accuracy, the main results are the following: (i) The rate constants k(on) and k(off) result in linear Arrhenius plots, consistent with the prediction of two-state association and dissociation. (ii) k(on) is independent of the nature of the base N located in the center of the third strand. (iii) k(on) strongly decreases when the NaCl concentration is decreased. (iv) The activation energy, E(on), is always negative and becomes more negative when the NaCl concentration is decreased. (v) k(off) is independent of NaCl concentration but depends on the base N, with its magnitude following the order C > G > A >> T. (vi) The activation energy, E(off), is independent of the base N. All these results are discussed in the light of a nucleation-zipping model similar to that developed for the duplex-coil transitions [Craig, M. E., Crothers, D. M., & Doty, P. (1971) J. Mol. Biol. 62,383-401; Porschke, D., Eigen, M. (1971) J. Mol. Biol. 62, 361-381].
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页码:9269 / 9278
页数:10
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