The Isolated RNase H Domain of Murine Leukemia Virus Reverse Transcriptase
The Isolated RNase H Domain of Murine Leukemia Virus Reverse Transcriptase
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DOI：
10.1074/jbc.272.35.22023
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发表时间：
1997-08
期刊：
影响因子：
--
通讯作者：
X. Zhan;R. Crouch
中科院分区：
文献类型：
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作者：
X. Zhan;R. Crouch
逆转录病毒RNA酶H在序列和结构上与大肠杆菌RNA酶HI相似，但在底物特异性、金属离子要求和比活性方面存在差异。将逆转录酶（RT）分离成聚合酶和RNase H结构域，从鼠白血病病毒（MuLV）中产生活性RNase H，但产生无活性的人类免疫缺陷病毒（HIV）RNase H。E. ColiRNase HI（但HIV RNase H中不存在）有助于与其底物结合，当插入HIV RNase H时，导致活性酶保留一定程度的特异性。在这里，我们展示了MuLV蛋白，其C末端含有175个氨基酸，具有自己的柄区或E. coliRNaseHI与RT的RNaseH具有相同的比活，保留了对Mn ~（2+）的选择性，结合率（KA）为E. coli RNase HI。然而，对于模型底物，去除tRNAPro引物的特异性和多嘌呤段稳定性丧失，表明MuLV的RNA酶H的特异性需要RT的剩余部分。KA的差异虽然显著，但似乎不足以解释细菌和病毒RNA酶H的特异性活性的差异。
Retroviral RNases H are similar in sequence and structure to Escherichia coli RNase HI and yet have differences in substrate specificities, metal ion requirements, and specific activities. Separation of reverse transcriptase (RT) into polymerase and RNase H domains yields an active RNase H from murine leukemia virus (MuLV) but an inactive human immunodeficiency virus (HIV) RNase H. The “handle region” present in E. coliRNase HI but absent in HIV RNase H contributes to the binding to its substrate and when inserted into HIV RNase H results in an active enzyme retaining some degree of specificity. Here, we show MuLV protein containing the C-terminal 175 amino acids with its own handle region or that of E. coli RNase HI has the same specific activity as the RNase H of RT, retains a preference for Mn2+ as the cation required for activity, and has association rate (K A ) 10% that of E. coli RNase HI. However, with model substrates, specificities for removal of the tRNAPro primer and polypurine tract stability are lost, indicating specificity of RNase H of MuLV requires the remainder of the RT. Differences in K A , while significant, appear insufficient to account for the differences in specific activities of the bacterial and viral RNases H.