Otsuka Yuichi, Koga Mitsunori, Iwamoto Akira, Yonesaki Tetsuro
Department of Biological Sciences, Graduate School of Science, Osaka University, Osaka, Japan.
Genes Genet Syst. 2007 Aug;82(4):291-9. doi: 10.1266/ggs.82.291.
Escherichia coli ribonuclease LS is a potential antagonist of bacteriophage T4. When the T4 dmd gene is defective, RNase LS cleaves T4 mRNAs and antagonizes T4 reproduction. Our previous work demonstrated that E. coli rnlA is essential for RNase LS activity. Here we show that His-tagged RnlA cleaves T4 soc RNA at one of the sites also cleaved by RNase LS in a cell extract. The cleavage activities of His-tagged RnlA and the RNase LS activity in a cell extract were inhibited by Dmd encoded by T4 phage. Fractionation of the RNase LS activity in a cell extract showed that it sedimented through a sucrose density gradient as a 1000-kDa complex that included RnlA. Pull-down experiments revealed more than 10 proteins associated with His-tagged RnlA. Among these, triose phosphate isomerase exhibited a remarkable affinity to RnlA. These results suggest that RnlA plays a central role in RNase LS activity and that its activity is regulated by multiple components.
大肠杆菌核糖核酸酶LS是噬菌体T4的潜在拮抗剂。当T4 dmd基因有缺陷时，核糖核酸酶LS会切割T4 mRNA并拮抗T4的繁殖。我们之前的工作表明，大肠杆菌rnlA对核糖核酸酶LS的活性至关重要。在此我们表明，带有组氨酸标签的RnlA在细胞提取物中于核糖核酸酶LS切割的位点之一切割T4 soc RNA。带有组氨酸标签的RnlA的切割活性以及细胞提取物中的核糖核酸酶LS活性受到T4噬菌体编码的Dmd的抑制。对细胞提取物中核糖核酸酶LS活性进行分级分离显示，它以包含RnlA的1000 kDa复合物的形式通过蔗糖密度梯度沉降。下拉实验揭示了10多种与带有组氨酸标签的RnlA相关的蛋白质。其中，磷酸丙糖异构酶对RnlA表现出显著的亲和力。这些结果表明，RnlA在核糖核酸酶LS活性中起核心作用，并且其活性受多种成分调节。
