Mutating a region of HIV-1 reverse transcriptase implicated in tRNALys-3 binding and the consequences for (-)-strand DNA synthesis
Mutating a region of HIV-1 reverse transcriptase implicated in tRNALys-3 binding and the consequences for (-)-strand DNA synthesis
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DOI：
10.1074/jbc.273.23.14523
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发表时间：
1998-06-05
影响因子：
4.8
通讯作者：
Le Grice, SFJ
中科院分区：
文献类型：
--
作者：
Arts, EJ;Miller, JT;Le Grice, SFJ
最近，tRNA（Lys-3）通过其反密码子环与人类免疫缺陷病毒I型（HIV-1）逆转录酶（RT）在230和357残基之间交联（Mishima， Y.和Steitz， J. a . (1995) EMBO J. 14, 2679-2687），扫描该区域表面，在引物-模板结合间隙外p66 thumb亚结构域的一个小缝隙两侧鉴定了三个基本氨基酸Lys(249), Arg（307）和Lys（311）。为了评估该区域与tRNA反密码子环的相互作用，这些p66残基被改变为Glu或Gin。p66亚基含有K249Q、K311Q、K311E和双R307E/K311E突变，与野生型p51形成稳定的二聚体。所有突变体对tRNA（Lys-3）的亲和力都降低，从该引物合成的（-）链DNA明显少于亲本异源二聚体。相比之下，这些变异有效地从寡核苷酸引物合成HIV-1（-）链强链DNA，对RNase H活性的影响最小，保留了RNA/DNA杂交的核内溶解和定向切割。通过对1,10-菲罗啉-铜介导的裂解的敏感性，原位足迹检测了二元RT.tRNA（Lys-3）复合物的结构特征。与野生型RT不同，突变体p66(K311Q)/p51和p66(K311E)/p51不能保护tRNA反密码子结构域免受化学切割，这表明二元RT tRNA复合物的结构发生了重大变化。这些结果表明，HIV-1 RT的p66拇指亚结构域的缝隙支持与tRNA（Lys-3）反密码子环的相互作用，而tRNA（Lys-3）反密码子环对高效（-）链DNA合成至关重要。
Recently, tRNA(Lys-3) Was cross-linked via its anticodon loop to human immunodeficiency virus type I (HIV-1) reverse transcriptase (RT) between residues 230 and 357 (Mishima, Y., and Steitz, J. A. (1995) EMBO J. 14, 2679-2687), Scanning the surface of this region identified three basic amino acids Lys(249), Arg(307), and Lys(311) flanking a small crevice on the p66 thumb subdomain outside the primer-template binding cleft. To assess an interaction of this region with the tRNA anticodon loop, these p66 residues were altered to Glu or Gin. p66 subunits containing K249Q, K311Q, K311E, and a dual R307E/K311E K311E mutation formed a stable dimer with wild type p51. All mutants showed reduced affinity for tRNA(Lys-3) and supported significantly less (-)-strand DNA synthesis from this primer than the parental heterodimer. In contrast, these variants efficiently synthesized HIV-1 (-)-strand strong-stop DNA from oligonucleotide primers and had minimal effect on RNase H activity, retaining endonucleolytic and directed cleavage of an RNA/DNA hybrid. Structural features of binary RT.tRNA(Lys-3) complexes were examined by in situ footprinting, via susceptibility to 1,10-phenanthroline-copper-mediated cleavage. Unlike wild type RT, mutants p66(K311Q)/p51 and p66(K311E)/p51 failed to protect the tRNA anticodon domain from chemical cleavage, indicating a significant structural alteration in the binary RT tRNA complex. These results suggest a crevice in the p66 thumb subdomain of HIV-1 RT supports an interaction with the tRNA(Lys-3) anticodon loop critical for efficient (-)-strand DNA synthesis.