RNASE III MECHANISM OF DOUBLE STRANDED RNA CLEAVAGE

RNA分子中的双链（ds）区域提供靶点， 由细胞核酸酶进行的分裂，这些核酸酶参与细胞的成熟和衰变 细胞和病毒RNA。 该项目的长期目标是 确定dsRNA的酶促切割机制及其在 调节细胞和病毒RNA的功能和代谢。 我们有 开发了RNase III模型，其中dsRNA结合基序 通过增强的结合刺激切割，也许还通过 活性酶二聚体的形成。 RNase III活性受到调节 通过丝氨酸的磷酸化，由噬菌体T7催化， 蛋白激酶 T7蛋白激酶也经历 自磷酸化伴随活性下调。 RNase III在至少两个丝氨酸上被T7蛋白磷酸化 激酶，其导致催化活性增加4倍。 知道 磷酸化位点，结合酶促测定， dsRNA结合和切割，将提供新的信息，如何 磷酸化调节RNA加工酶。 的 T7在一个或多个丝氨酸残基上的自磷酸化也是 兴趣 然后可以将鉴定的磷酸丝氨酸改变为 丙氨酸，产生抗自磷酸化蛋白激酶 其可表现出持续的磷酸化活性。 未来 生物化学研究将确定其他细胞靶点， 通过T7蛋白激酶的磷酸化。

Double-stranded (ds) regions in RNA molecules provide targets for cleavage by cellular nucleases involved in the maturation and decay of cellular and viral RNAs. The long-range goal of this project is to determine the mechanism of enzymatic cleavage of dsRNA and its role in regulating cellular and viral RNA function and metabolism. We have developed an RNase III model wherein the dsRNA-binding motif stimulates cleavage through enhanced binding, and perhaps also through formation of the active enzyme dimer. RNase III activity is regulated by phosphorylation on serine, catalyzed by the bacteriophage T7 protein kinase. The T7 protein kinase also undergoes autophosphorylation with concomitant down-regulation of activity. RNase III is phosphorylated on at least two serines by the T7 protein kinase, which causes a 4-fold increase in catalytic activity. Knowing the sites of phosphorylation, in conjunction with enzymatic assays for dsRNA binding and cleavage, will provide new information on how phosphorylation regulates an RNA processing enzyme. The autophosphorylation of T7 on one or more serine residues is also of interest. The identified phosphoserines can then be changed to alanine, creating an autophosphorylation-resistant protein kinase which may exhibit sustained phosphorylation activity. Future biochemical studies will determine the other cellular targets of phosphorylation by the T7 protein kinase.