Tokumoto Y, Saigo K
Department of Biophysics and Biochemistry, Faculty of Science, University of Tokyo, Japan.
Nucleic Acids Symp Ser. 1992(27):21-2.
The sTobRV(+) ribozyme consists of a small catalytic domain and two wing sequences(1). By changing its wing sequences, the ribozyme can cleave many different RNAs in a site-specific manner, functioning as an RNA restriction enzyme(1). Although relatively strong ligase activity is known to be associated with sTobRV(+) RNA(2,3), the sTobRV(+) ribozyme itself has been claimed to have no ligase activity. Here, we show the evidence that the sTobRV(+) ribozyme has the ability to rejoin its digestion products at low temperatures such as 4 degrees C. In contrast, little or no ligation product can be produced at 50 degrees C, the temperature giving the maximum digestion activity. The ligation reaction requires Mg++ ion. The first substrate (P1, see Fig.1), possessing 2',3' cyclic phosphate, must be RNA, but the second substrate (P2), required to have 5'OH, can be replaced by DNA counterparts, equal to or longer than dimer, thus making it possible to generate RNA-DNA chimeric molecules. We also show the resultant RNA-DNA chimera to be digestable by the sTobRV(+) ribozyme. RNase digestion indicates the phosphodiester linkage thus generated to be exclusively 3'-5'.
sTobRV(+)核酶由一个小的催化结构域和两个侧翼序列组成(1)。通过改变其侧翼序列，该核酶能够以位点特异性方式切割许多不同的RNA，起到RNA限制酶的作用(1)。尽管已知相对较强的连接酶活性与sTobRV(+) RNA相关(2,3)，但sTobRV(+)核酶本身据称没有连接酶活性。在此，我们展示了证据，表明sTobRV(+)核酶在诸如4℃的低温下有能力重新连接其消化产物。相比之下，在50℃（产生最大消化活性的温度）几乎不产生或不产生连接产物。连接反应需要Mg++离子。第一个底物（P1，见图1）具有2',3'环磷酸，必须是RNA，但第二个底物（P2）需要有5'OH，可以被等于或长于二聚体的DNA对应物替代，从而有可能产生RNA-DNA嵌合分子。我们还展示了所得的RNA-DNA嵌合体可被sTobRV(+)核酶消化。核糖核酸酶消化表明由此产生的磷酸二酯键完全是3'-5'的。
