Mg^(2+)和Na^+对多核酶系统体外切割反应的影响(英文)

Effects of Divalent Mg^(2+) and Monovalent Na^+ on Cleavage Reactions by Multiribozyme System in vitro

为了进一步了解2价Mg2+和1价Na+存在与否的情况下,多核酶系统对底物RNA的切割效率,构建了pGEM-Coat′A,pGEM-Coat′A196Rz质粒和pGEM-MDR1靶质粒.通过用SP6/T7转录试剂盒在体外转录RNA,在无细胞系统进行切割反应,反应产物通过6%变性聚丙烯酰胺凝胶电泳,干胶、X光片曝光自显影,利用ImageJ生物图像分析软件分析.结果表明,多核酶系统的切割效率依赖于二价Mg2+的浓度,切割产物随Mg2+浓度的增加而增加,而且具有反应时间的依赖性.在Na+浓度低于200mmol/L且单独存在时,没有切割产物生成.相反,在Na+和Mg2+共存时,表现出Na+抑制Mg2+诱导的切割活性,切割效率明显低于Mg2+单独存在时的结果.这些结果提示,在生理环境下,Mg2+对于多核酶系统对底物的切割反应是必需的,而Na+则不是.

To better understand the cleavage efficiency of multiribozyme system on its RNA substrate in the presence and absence of divalent magnesium and monovalent sodium ions, we constructed pGEM-Coat＇ A, pGEM-Coat＇A196Rz plasmids and pGEM-MDR1 target plasmid. They were applied to transcribe RNAs with SP6/T7 transcription kit. Cleavage reactions were carried out in cell-free system and reaction products were analyzed by electrophoresis on 6% denaturing polyacrylamide gels in TBS buffer. The gels were dried and exposed to X-ray films for autoradiography. The Image J software was employed to analyze the dried gels. The results indicated that the cleavage efficiency of the multiribozyme was dependent on the concentration of divalent Mg^2＋ . The cleavage products increased with the concentrations of divalent Mg^2＋ and were Mg^2＋- concentration and time dependent. No cleavage product was obtained in the presence of lower than 200 mmol/L Na^＋ alone. On the contrary, monovalent Na^＋ inhibited the Mg^2＋ -induced cleavage reaction in Na^＋ and Mg^2＋ coexistance. The cleavage rate was significantly lower than that observed with divalent Mg^2＋ alone. These results suggested that divalent Mg^2＋ was required for multiribozyme on substrate cleavage reaction in the physical condition, whereas monovalent Na^＋ was not.