Centre for Cellular and Molecular Biology, Uppal Road, Hyderabad 500007, India.
Appl Environ Microbiol. 2011 Nov;77(22):7896-904. doi: 10.1128/AEM.05683-11. Epub 2011 Sep 16.
RNase R is a highly processive, hydrolytic 3'-5' exoribonuclease belonging to the RNB/RNR superfamily which plays significant roles in RNA metabolism in bacteria. The enzyme was observed to be essential for growth of the psychrophilic Antarctic bacterium Pseudomonas syringae Lz4W at a low temperature. We present results here pertaining to the biochemical properties of RNase R and the RNase R-encoding gene (rnr) locus from this bacterium. By cloning and expressing a His₆-tagged form of the P. syringae RNase R (RNase R(Ps)), we show that the enzyme is active at 0 to 4°C but exhibits optimum activity at ∼25°C. The enzyme is heat labile in nature, losing activity upon incubation at 37°C and above, a hallmark of many psychrophilic enzymes. The enzyme requires divalent cations (Mg²⁺ and Mn²⁺) for activity, and the activity is higher in 50 to 150 mM KCl when it largely remains as a monomer. On synthetic substrates, RNase R(Ps) exhibited maximum activity on poly(A) and poly(U) in preference over poly(G) and poly(C). The enzyme also degraded structured malE-malF RNA substrates. Analysis of the cleavage products shows that the enzyme, apart from releasing 5'-nucleotide monophosphates by the processive exoribonuclease activity, produces four-nucleotide end products, as opposed to two-nucleotide products, of RNA chain by Escherichia coli RNase R. Interestingly, three ribonucleotides (ATP, GTP, and CTP) inhibited the activity of RNase R(Ps) in vitro. The ability of the nonhydrolyzable ATP-γS to inhibit RNase R(Ps) activity suggests that nucleotide hydrolysis is not required for inhibition. This is the first report on the biochemical property of a psychrophilic RNase R from any bacterium.
核糖核酸酶 R 是一种高度连续的、水解 3'-5'外切核酸酶，属于 RNB/RNR 超家族，在细菌的 RNA 代谢中发挥重要作用。该酶被观察到在低温下对嗜冷南极假单胞菌 Lz4W 的生长是必需的。我们在此介绍了来自该细菌的核糖核酸酶 R（RNase R）和 RNase R 编码基因（rnr）位点的生化特性的结果。通过克隆和表达 His₆ 标记的假单胞菌 RNase R（RNase R(Ps)）形式，我们表明该酶在 0 至 4°C 下具有活性，但在约 25°C 下表现出最佳活性。该酶本质上是热不稳定的，在 37°C 及以上孵育时会失去活性，这是许多嗜冷酶的特征。该酶需要二价阳离子（Mg²⁺和 Mn²⁺）才能发挥活性，在 50 至 150 mM KCl 中活性更高，此时它主要保持单体形式。在合成底物上，RNase R(Ps) 对 poly(A) 和 poly(U) 的活性最高，优先于 poly(G) 和 poly(C)。该酶还降解结构型 malE-malF RNA 底物。对切割产物的分析表明，除了通过连续外切核酸酶活性释放 5'-核苷酸单磷酸外，该酶还产生四核苷酸末端产物，而不是大肠杆菌 RNase R 产生的 RNA 链的二核苷酸产物。有趣的是，三种核苷酸（ATP、GTP 和 CTP）在体外抑制 RNase R(Ps) 的活性。非水解型 ATP-γS 抑制 RNase R(Ps) 活性表明核苷酸水解不是抑制所必需的。这是首次报道来自任何细菌的嗜冷 RNase R 的生化特性。
