Burke D H, Scates L, Andrews K, Gold L
Department of Molecular, Cellular and Development Biology, University of Colorado, Boulder 80309-0347, USA.
J Mol Biol. 1996 Dec 13;264(4):650-66. doi: 10.1006/jmbi.1996.0667.
The reverse transcriptase (RT) of the human immunodeficiency virus (HIV) is a proven target for therapeutic intervention of HIV infections. We have found several new RNA inhibitors of HIV-1 RT that differ significantly from the pseudoknot ligands found previously, along with a wide variety of pseudoknot variants. One pseudoknot variant and three novel ligands were studied in more detail. Each specifically inhibits DNA polymerization by HIV RT (half-maximal inhibition at 0.3 to 20 nM inhibitor), but not that of RTs derived from MMLV or AMV. The minimal binding element of each isolate was determined by deletion analysis and by gel electrophoresis of protein-bound, partially alkaline-hydrolyzed RNA. Truncations of three of the isolates bound nearly as well as (or better than) the parental sequences, while most deletions in the fourth caused substantial disruption of binding. The truncated versions of two isolates were subjected to six rounds of secondary SELEX after resynthesizing them mutagenically. Patterns of conserved and covarying nucleotides yielded structural models consistent with 5' and 3' boundary determinations for these molecules. Among the four isolates studied in detail, the first is confirmed as being a pseudoknot, albeit with substantial structural differences as compared to the canonical pseudoknots identified previously. The second forms a stem-loop structure with additional flanking sequences required for binding. Tentative structural models for the other two isolates are presented. The minimal fully active truncations of each of these four isolates compete with each other and with a classical RNA pseudoknot for binding to HIV RT, suggesting that they all recognize the same or overlapping sites on the protein, in spite of their apparently dissimilar structures. We model their interactions with RT as mimicking the 40 to 45 degrees bend in dsDNA co-crystallized with RT.
人类免疫缺陷病毒（HIV）的逆转录酶（RT）是HIV感染治疗干预的一个已证实的靶点。我们发现了几种新的HIV-1 RT的RNA抑制剂，它们与先前发现的假结配体有显著差异，同时还有各种各样的假结变体。对一种假结变体和三种新型配体进行了更详细的研究。每种抑制剂都能特异性抑制HIV RT的DNA聚合作用（在0.3至20 nM抑制剂浓度下达到半数最大抑制），但对源自莫洛尼鼠白血病病毒（MMLV）或禽成髓细胞瘤病毒（AMV）的RT没有抑制作用。通过缺失分析以及与蛋白质结合的、部分碱水解RNA的凝胶电泳，确定了每种分离物的最小结合元件。三种分离物的截短形式结合能力与亲本序列相近（或更好），而第四种分离物的大多数缺失则导致结合的大量破坏。对两种分离物的截短版本进行诱变重新合成后，进行了六轮二级指数富集的配体系统进化技术（SELEX）。保守和共变核苷酸模式产生的结构模型与这些分子的5'和3'边界测定结果一致。在详细研究的四种分离物中，第一种被确认为假结，尽管与先前鉴定的典型假结相比有很大的结构差异。第二种形成了一个茎环结构，结合需要额外的侧翼序列。给出了另外两种分离物的初步结构模型。这四种分离物各自最小的完全活性截短形式相互竞争，并与一种经典RNA假结竞争与HIV RT的结合，这表明尽管它们的结构明显不同，但它们都识别蛋白质上相同或重叠的位点。我们将它们与RT的相互作用模拟为与RT共结晶的双链DNA中40至45度的弯曲。
