Permanasari Etin-Diah, Yasukawa Kiyoshi, Kanaya Shigenori
Department of Material and Life Science, Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka, 565-0871, Japan.
Mol Biotechnol. 2015 Jun;57(6):526-38. doi: 10.1007/s12033-015-9846-5.
Thermotoga maritima RNase H1 and Bacillus stearothermophilus RNase H2 have an N-terminal substrate binding domain, termed hybrid binding domain (TmaHBD), and N-terminal domain (BstNTD), respectively. HIV-1 reverse transcriptase (RT) is a heterodimer consisting of a P66 subunit and a P51 subunit. The P66 subunit contains a C-terminal RNase H domain, which exhibits RNase H activity either in the presence of Mg(2+) or Mn(2+) ions. The isolated RNase H domain of HIV-1 RT (RNH(HIV)) is inactive, possibly due to the lack of a substrate binding ability, disorder of a loop containing His539, and increased flexibility. To examine whether the activity of RNH(HIV) is restored by the attachment of TmaHBD or BstNTD to its N-terminus, two chimeric proteins, TmaHBD-RNH(HIV) and BstNTD-RNH(HIV), were constructed and characterized. Both chimeric proteins bound to RNA/DNA hybrid more strongly than RNH(HIV) and exhibited enzymatic activity in the presence of Mn(2+) ions. They did not exhibit activity or exhibited very weak activity in the presence of Mg(2+) ions. These results indicate that TmaHBD and BstNTD function as an RNA/DNA hybrid binding tag, and greatly increase the substrate binding affinity and Mn(2+)-dependent activity of RNH(HIV) but do not restore the Mg(2+)-dependent activity of RNH(HIV).
嗜热栖热菌核糖核酸酶H1（Thermotoga maritima RNase H1）和嗜热脂肪芽孢杆菌核糖核酸酶H2（Bacillus stearothermophilus RNase H2）分别具有一个N端底物结合结构域，称为杂交结合结构域（TmaHBD）和N端结构域（BstNTD）。HIV-1逆转录酶（RT）是由一个P66亚基和一个P51亚基组成的异源二聚体。P66亚基包含一个C端核糖核酸酶H结构域，该结构域在Mg(2+)或Mn(2+)离子存在时表现出核糖核酸酶H活性。分离出的HIV-1 RT核糖核酸酶H结构域（RNH(HIV)）无活性，可能是由于缺乏底物结合能力、包含His539的环无序以及灵活性增加。为了研究将TmaHBD或BstNTD连接到RNH(HIV)的N端是否能恢复其活性，构建并表征了两种嵌合蛋白TmaHBD-RNH(HIV)和BstNTD-RNH(HIV)。两种嵌合蛋白与RNA/DNA杂交体的结合都比RNH(HIV)更强，并且在Mn(2+)离子存在时表现出酶活性。在Mg(2+)离子存在时，它们不表现出活性或表现出非常弱的活性。这些结果表明，TmaHBD和BstNTD作为RNA/DNA杂交结合标签发挥作用，并大大提高了RNH(HIV)对底物的结合亲和力和依赖Mn(2+)的活性，但不能恢复RNH(HIV)依赖Mg(2+)的活性。
