Kumar A, Kim H R, Sobol R W, Becerra S P, Lee B J, Hatfield D L, Suhadolnik R J, Wilson S H
Sealy Center for Molecular Science, University of Texas Medical Branch, Galveston 77555.
Biochemistry. 1993 Jul 27;32(29):7466-74. doi: 10.1021/bi00080a018.
Human immunodeficiency virus type-1 (HIV-1) reverse transcriptase (RT) and its domain fragments were used to map nucleic acid binding sites within the enzyme. Discrete domain fragments were produced after the digestion of three forms of RT (p66, p66/p51 heterodimer, and p51) with V8 protease or trypsin, and the primary structure of each domain fragment was mapped by both immunoblotting and N-terminal amino acid sequence analysis. These domain fragments represent N-terminal, middle, or C-terminal regions of RT. Using Northwestern or Southwestern blotting assays, the domain fragments were evaluated for nucleic acid binding. In this technique, RT proteins are electroblotted onto the membrane and renatured after SDS-PAGE; the proteins are then probed with the primer analogues 32P-labeled d(T)16 or 32P-labeled tRNA(Lys,3). A V8 protease domain fragment spanning residues 195 to approximately 300 (p12), which was found earlier to be UV cross-linked to the primer in intact RT [Sobol et al. (1991) Biochemistry 30, 10623-10631], showed binding to both nucleic acid probes. We first localized nucleic acid binding in p66 to an N-terminal domain fragment of residues 1 approximately equal to 300. By contrast, a C-terminal domain fragment termed p30(303 approximately equal to 560) did not show nucleic acid binding. To investigate the role of the region just N-terminal to residue 303, an expression vector named pRC-35 encoding residues 273-560 was constructed.(ABSTRACT TRUNCATED AT 250 WORDS)
人类免疫缺陷病毒1型（HIV-1）逆转录酶（RT）及其结构域片段被用于定位该酶内的核酸结合位点。用V8蛋白酶或胰蛋白酶消化三种形式的RT（p66、p66/p51异二聚体和p51）后产生了离散的结构域片段，并且通过免疫印迹和N端氨基酸序列分析对每个结构域片段的一级结构进行了定位。这些结构域片段代表RT的N端、中间或C端区域。使用蛋白质印迹或核酸印迹分析来评估结构域片段的核酸结合情况。在这项技术中，RT蛋白在SDS-PAGE后被电印迹到膜上并复性；然后用引物类似物32P标记的d(T)16或32P标记的tRNA(Lys,3)对蛋白进行检测。一个跨越残基195至约300的V8蛋白酶结构域片段（p12），之前发现在完整的RT中能与引物发生紫外线交联[Sobol等人（1991年）《生物化学》30，10623 - 10631]，显示出与两种核酸探针都有结合。我们首先将p66中的核酸结合定位到残基1至约300的N端结构域片段。相比之下，一个称为p30(303至约560)的C端结构域片段未显示出核酸结合。为了研究残基303紧邻的N端区域的作用，构建了一个编码残基273 - 560的表达载体pRC - 35。（摘要截短于250词）
