Nishimura Kosaku, Yokokawa Kanta, Hisayoshi Tetsuro, Fukatsu Kosuke, Kuze Ikumi, Konishi Atsushi, Mikami Bunzo, Kojima Kenji, Yasukawa Kiyoshi
Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University, Sakyo-ku, Kyoto 606-8502, Japan.
Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Gokasho, Uji, Kyoto 611-0011, Japan.
Protein Expr Purif. 2015 Sep;113:44-50. doi: 10.1016/j.pep.2015.04.012. Epub 2015 May 7.
Moloney murine leukemia virus reverse transcriptase (MMLV RT) contains fingers, palm, thumb, and connection subdomains as well as an RNase H domain. The DNA polymerase active site resides in the palm subdomain, and the RNase H active site is located in the RNase H domain. The RNase H domain contains a positively charged α-helix called the C helix (H(594)GEIYRRR(601)), that is thought to be involved in substrate recognition. In this study, we expressed three versions of the RNase H domain in Escherichia coli, the wild-type domain (WT) (residues Ile498-Leu671) and two variants that lack the regions containing the C helix (Ile593-Leu603 and Gly595-Thr605, which we called ΔC1 and ΔC2, respectively) with a strep-tag at the N-terminus and a deca-histidine tag at the C-terminus. These peptides were purified from the cells by anion-exchange, Ni(2+) affinity, and Strep-Tactin affinity column chromatography, and then the tags were removed by proteolysis. In an RNase H assay using a 25-bp RNA-DNA heteroduplex, WT, ΔC1, and ΔC2 produced RNA fragments ranging from 7 to 16 nucleotides (nt) whereas the full-length MMLV RT (Thr24-Leu671) produced 14-20-nt RNA fragments, suggesting that elimination of the fingers, palm, thumb, and connection subdomains affects the binding of the RNase H domain to the RNA-DNA heteroduplex. The activity levels of WT, ΔC1, and ΔC2 were estimated to be 1%, 0.01%, and 0.01% of full-length MMLV RT activity, indicating that the C helix is important, but not critical, for the activity of the isolated RNase H domain.
莫洛尼鼠白血病病毒逆转录酶（MMLV RT）包含指状结构域、掌状结构域、拇指结构域、连接结构域以及一个核糖核酸酶H结构域。DNA聚合酶活性位点位于掌状结构域，核糖核酸酶H活性位点位于核糖核酸酶H结构域。核糖核酸酶H结构域包含一个带正电荷的α螺旋，称为C螺旋（H(594)GEIYRRR(601)），被认为参与底物识别。在本研究中，我们在大肠杆菌中表达了核糖核酸酶H结构域的三个版本，即野生型结构域（WT）（第498位异亮氨酸至第671位亮氨酸）和两个缺失包含C螺旋区域的变体（第593位异亮氨酸至第603位亮氨酸和第595位甘氨酸至第605位苏氨酸，我们分别称之为ΔC1和ΔC2），在N端带有链霉亲和标签，在C端带有十组氨酸标签。这些肽通过阴离子交换、镍（2+）亲和和链霉亲和素亲和柱色谱从细胞中纯化出来，然后通过蛋白酶解去除标签。在使用25碱基对RNA-DNA杂交双链体的核糖核酸酶H测定中，WT、ΔC1和ΔC2产生了7至16个核苷酸（nt）的RNA片段，而全长MMLV RT（第24位苏氨酸至第671位亮氨酸）产生了14至20个核苷酸的RNA片段，这表明去除指状结构域、掌状结构域、拇指结构域和连接结构域会影响核糖核酸酶H结构域与RNA-DNA杂交双链体的结合。WT、ΔC1和ΔC2的活性水平估计分别为全长MMLV RT活性的1%、0.01%和0.01%，表明C螺旋对于分离的核糖核酸酶H结构域的活性很重要，但并非关键因素。
