Cerritelli S M, Crouch R J
Laboratory of Molecular Genetics, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20892, USA.
RNA. 1995 May;1(3):246-59.
Eukaryotic ribonucleases H of known sequence are composed of an RNase H domain similar in size and sequence to that of Escherichia coli RNase HI and additional domains of unknown function. The RNase H1 of Saccharomyces cerevisiae has such an RNase H domain at its C-terminus. Here we show that the N-terminal non-RNase H portion of the yeast RNase H1 binds tightly to double-stranded RNA (dsRNA) and RNA-DNA hybrids even in the absence of the RNase H domain. Two copies of a sequence with limited similarity to the dsRNA-binding motif are present in this N-terminus. When the first of these sequences is altered, the protein no longer binds tightly to dsRNA and exhibits an increase in RNase H activity. Unlike other dsRNA-binding proteins, increasing the Mg2+ concentration from 0.5 mM to 5 mM inhibits binding of RNase H1 to dsRNA; yet a protein missing the RNase H domain binds strongly to dsRNA even at the higher Mg2+ concentration. These results suggest that binding to dsRNA and RNase H activity are mutually exclusive, and the Mg2+ concentration is critical for switching between the activities. Changes in the Mg2+ concentration or proteolytic severing of the dsRNA-binding domain could alter the activity or location of the RNase H and may govern access of the enzyme to the substrate. Sequences similar to the dsRNA-binding motif are present in other eukaryotic RNases H and the transactivating protein of cauliflower mosaic virus, suggesting that these proteins may also bind to dsRNA.
已知序列的真核核糖核酸酶H由一个与大肠杆菌核糖核酸酶HI大小和序列相似的核糖核酸酶H结构域以及其他功能未知的结构域组成。酿酒酵母的核糖核酸酶H1在其C端有这样一个核糖核酸酶H结构域。我们在此表明，酵母核糖核酸酶H1的N端非核糖核酸酶H部分即使在没有核糖核酸酶H结构域的情况下也能紧密结合双链RNA（dsRNA）和RNA-DNA杂交体。在这个N端存在两个与dsRNA结合基序相似度有限的序列拷贝。当其中第一个序列发生改变时，该蛋白不再紧密结合dsRNA，并且核糖核酸酶H活性增加。与其他dsRNA结合蛋白不同，将Mg2+浓度从0.5 mM提高到5 mM会抑制核糖核酸酶H1与dsRNA的结合；然而，一个缺失核糖核酸酶H结构域的蛋白即使在较高的Mg2+浓度下也能与dsRNA强烈结合。这些结果表明，与dsRNA的结合和核糖核酸酶H活性是相互排斥 的，并且Mg2+浓度对于在这些活性之间切换至关重要。Mg2+浓度的变化或dsRNA结合结构域的蛋白水解切割可能会改变核糖核酸酶H的活性或位置，并可能控制该酶对底物的作用。与dsRNA结合基序相似的序列存在于其他真核核糖核酸酶H和花椰菜花叶病毒的反式激活蛋白中，这表明这些蛋白也可能与dsRNA结合。
