BIOCHEMICAL ANALYSIS OF HIV REVERSE TRANSCRIPTASE AND REV PROTEIN FUNCTION

两种不同类型的交联方法用于绘图的功效 复杂大分子中特异性RNA-RNA和RNA-蛋白质接触 最近，该组织被证实。 这些将被用于获得 深入了解两种HIV编码蛋白的作用机制 其结构正在由本程序的其他成员进行分析： 逆转录酶（RT）和Rev蛋白。 HIV RT的结构最近被解析为3.2埃 分辨率为T。史泰兹的实验室。 模型构建表明， 人tRNA 3Lys引物可能与p66和p51亚基相互作用， 其3'端在聚合酶活性位点与基因组RNA杂交 template. 为了验证所提出的tRNA-酶相互作用，一系列 将合成含有单个4- 硫尿苷前面是单个32 P-磷酸基团。 孵育后 这些tRNA与逆转录酶发生转录反应， 在> 300 nm的光照射下特异性地激活4-thiouU，产生 RNA-蛋白质交联。 用P1核酸酶完全消化（其产生 5'磷酸）然后将标记转移到RT亚基。 曾经各种 tRNA中的位置已被鉴定为与p66或p51 亚基，分析可以扩展到映射交联肽/氨基 酸，并且还绘制模板-RT交联。 HIV Rev蛋白已被提议促进未剪接的 通过解离部分组装的含有rre结合位点的RNA 剪接体，主要基于体外系统的结果，其中非常 必须使用更高浓度的Rev来抑制剪接。 的 Rev对体内剪接体组装的影响将使用 果蝇组织培养系统（SL 2细胞），其表现出Rev依赖性 从诱导型启动子表达的env RNA的输出。 在体内-peptien 交联将鉴定哪些snRNA与杂交选择的 在不存在和存在Rev的情况下，来自细胞的env RNA。 探针选择snRNAs，允许检测剪接依赖的 前体mRNA底物与U1、U2、U 5、U6和U2/U6 snRNA之间的交联 以及开发用于绘制交联位点的方法。 类似 对体内env RNA-snRNA交联的分析应该可以确定 此时Rev作用于终止剪接体组装。

The efficacy of two different types of crosslinking approaches for mapping specific RNA-RNA and RNA-protein contacts in complex macromolecular assemblies has recently been demonstrated. These will be applied to gain insight into the mechanism of action of two of the HIV-encoded proteins whose structures are under analysis by other members of this program: the reverse transcriptase (RT) and the Rev protein. The structure of the HIV RT has recently been solved to 3.2angstroms resolution in T. Steitz's laboratory. Model building suggests how the human tRNA3Lys primer might interact with the p66 and p51 subunits, placing its 3' end in the polymerase active site hybridized to the genomic RNA template. To verify the proposed tRNA-enzyme interactions, a series of tRNA3Lys transcripts will be synthesized which contain a single 4- thiouridine preceded by a single 32P-phosphate group. After incubation of these tRNAs with reverse transcriptase in transcription reactions, irradiation at > 300nm light specifically activates the 4-thioU, generating RNA-protein crosslinks. Complete digestion with P1 nuclease (which creates 5' phosphates) then transfers label to the RT subunits. Once various positions in the tRNA have been identified as contacting the p66 or p51 subunit, the analyses can be extended to map crosslinked peptides/amino acids, and also to map template-RT crosslinks. The HIV Rev protein has been proposed to facilitate the export of unspliced RNAs containing the rre binding site by dissociating partially assembled spliceosomes, based primarily on results from in vitro systems, where very high concentrations of Rev must be utilized to inhibit splicing. The effect of Rev on spliceosome assembly in vivo will be examined using a Drosophila tissue culture system (SL2 cells) which exhibits Rev-dependent export of env RNA expressed from an inducible promoter. In vivo-psoralen crosslinking will identify which snRNAs are associated with hybrid-selected env RNA from cells in the absence and presence of Rev. Use of specific probes to select snRNAs, has allowed detection of splicing-dependent crosslinks between a pre-mRNA substrate and U1,U2,U5,U6 and U2/U6 snRNAs and the development of methods for mapping the crosslinked sites. Similar analyses of the in vivo env RNA-snRNA crosslinks should identify the point at which Rev acts to abort spliceosome assembly.