Sawata S, Shimayama T, Komiyama M, Kumar P K, Nishikawa S, Taira K
Department of Industrial Chemistry, Faculty of Engineering, University of Tokyo, Japan.
Nucleic Acids Res. 1993 Dec 11;21(24):5656-60. doi: 10.1093/nar/21.24.5656.
In order to characterize structure-function relationships, the kinetic behavior of chimeric RNA/DNA ribozyme was compared with that of all RNA ribozyme. Determined kcat values were proven to represent the chemical-cleavage step and not the product-dissociation step. In agreement with the finding by Dahm and Uhlenbeck [Biochemistry 30, 9464-9469 (1991)], various metal ions, including Co2+ and Ca2+ with the ionic radius of 0.65 and 1.0 A, respectively, could support hammerhead cleavage for both types of ribozyme. Measurements of kinetic parameters in the presence of various divalent metal ions revealed that DNA arms always enhanced kcat values. Chemical-probing data using dimethylsulfate indicated that the catalytic-loop structures of all-RNA and chimeric ribozymes were nearly identical with the exception of enhanced termination of primer extension reactions at C3 in the case of the chimeric ribozyme. These observations and others demonstrate that DNA substitution in non-catalytic-loop regions increases chemical-cleavage activity, possibly with an accompanying very subtle change in the structure.
为了表征结构-功能关系，将嵌合RNA/DNA核酶的动力学行为与全RNA核酶的动力学行为进行了比较。已证实所测定的kcat值代表化学切割步骤而非产物解离步骤。与达姆和乌伦贝克的研究结果一致[《生物化学》30, 9464 - 9469(1991)]，各种金属离子，包括离子半径分别为0.65 Å和1.0 Å的Co2+和Ca2+，都能支持这两种类型核酶的锤头状切割。在各种二价金属离子存在下对动力学参数的测量表明，DNA臂总是能提高kcat值。使用硫酸二甲酯的化学探针数据表明，除了嵌合核酶在C3处引物延伸反应的终止增强外，全RNA核酶和嵌合核酶的催化环结构几乎相同。这些观察结果及其他结果表明，非催化环区域的DNA替代增加了化学切割活性，结构可能伴随非常细微的变化。
