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

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.