Department of Biochemistry, Robert Wood Johnson Medical School, UMDNJ, CABM, 679 Hoes Lane, Piscataway, New Jersey 08854, USA.
J Bacteriol. 2010 Mar;192(5):1344-52. doi: 10.1128/JB.01368-09. Epub 2009 Dec 18.
In Escherichia coli, the cold shock response occurs when there is a temperature downshift from 37 degrees C to 15 degrees C, and this response is characterized by induction of several cold shock proteins, including the DEAD-box helicase CsdA, during the acclimation phase. CsdA is involved in a variety of cellular processes. Our previous studies showed that the helicase activity of CsdA is critical for its function in cold shock acclimation of cells and that the only proteins that were able to complement its function were another helicase, RhlE, an RNA chaperone, CspA, and a cold-inducible exoribonuclease, RNase R. Interestingly, other major 3'-to-5' processing exoribonucleases of E. coli, such as polynucleotide phosphorylase and RNase II, cannot complement the cold shock function of CsdA. Here we carried out a domain analysis of RNase R and showed that this protein has two distinct activities, RNase and helicase, which are independent of each other and are due to different domains. Mutant RNase R proteins that lack the RNase activity but exhibit the helicase activity were able to complement the cold shock function of CsdA, suggesting that only the helicase activity of RNase R is essential for complementation of the cold shock function of CsdA. We also observed that in vivo deletion of the two cold shock domains resulted in a loss of the ability of RNase R to complement the cold shock function of CsdA. We further demonstrated that RNase R exhibits helicase activity in vitro independent of its RNase activity. Our results shed light on the unique properties of RNase R and how it is distinct from other exoribonucleases in E. coli.
在大肠杆菌中，当温度从 37°C 下降到 15°C 时，会发生冷休克反应，在适应阶段，会诱导几种冷休克蛋白，包括 DEAD-box 解旋酶 CsdA。CsdA 参与多种细胞过程。我们之前的研究表明，CsdA 的解旋酶活性对于其在细胞冷休克适应中的功能至关重要，并且唯一能够补充其功能的蛋白质是另一种解旋酶 RhlE、RNA 伴侣 CspA 和冷诱导的外切核酸酶 RNase R。有趣的是，大肠杆菌的其他主要 3'到 5'加工外切核酸酶，如多核苷酸磷酸化酶和 RNase II，不能补充 CsdA 的冷休克功能。在这里，我们对 RNase R 进行了结构域分析，表明该蛋白具有两种不同的活性，即 RNase 和解旋酶，它们彼此独立，并且由不同的结构域组成。缺乏 RNase 活性但表现出解旋酶活性的突变 RNase R 蛋白能够补充 CsdA 的冷休克功能，表明只有 RNase R 的解旋酶活性对于补充 CsdA 的冷休克功能是必需的。我们还观察到，体内缺失两个冷休克结构域会导致 RNase R 丧失补充 CsdA 的冷休克功能的能力。我们进一步证明，RNase R 在体外独立于其 RNase 活性表现出解旋酶活性。我们的研究结果揭示了 RNase R 的独特性质以及它与大肠杆菌中其他外切核酸酶的区别。
