BIOCHEMICAL ANALYSIS OF HIV REVERSE TRANSCRIPTASE AND REV PROTEIN FUNCTION

两种不同类型的交联法作图的效果 复杂大分子中特定的RNA-RNA和RNA-蛋白质接触 组件最近已被演示。这些将应用于获得 深入了解两种HIV编码蛋白的作用机制 该计划的其他成员正在分析其结构： 逆转录酶(RT)和REV蛋白。 HIV RT的结构最近被解决到3.2埃 在T.Steitz的实验室中的解决方案。模型构建表明了 人tRNA3Lys引物可能与p66和p51亚基相互作用，将 它在聚合酶活性部位的3‘端与基因组RNA杂交 模板。为了验证所提出的tRNA-酶相互作用，一系列 TRNA3Lys转录本将被合成，它包含一个单一的4- 硫代尿苷前面有一个32P-磷酸基团。在孵化了 这些在转录反应中带有逆转录酶的tRNA， 300 nm的光照射可以特异性地激活4-硫代，产生 RNA-蛋白质的交联物。用P1核酸酶完全消化(这会产生 5‘磷酸盐)然后将标记转移到RT亚基。曾经多种多样 TRNA中的位置已被确定为与p66或p51相联系 亚基，这种分析可以扩展到定位交联肽/氨基 酸，也作图模板-RT交联物。 HIV Rev蛋白已被提议用于促进未剪接体的出口 通过解离部分组装而含有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 必须使用高浓度的REV来抑制剪接。这个 REV对体内剪接体组装的影响将通过 果蝇组织培养系统(SL2细胞)表现出REV依赖性 从可诱导启动子表达的env RNA的输出。体内-补骨脂素 交联会识别哪些SnRNA与杂交选择的SnRNA关联 来自细胞的env RNA在没有和存在Rev.的情况下特异性地使用 用于选择SnRNA的探针，允许检测依赖剪接的 前信使核糖核酸底物与U1、U2、U5、U6和U2/U6单链RNA的交联性 以及绘制交联点图谱的方法。类似 对活体包膜RNA-SnRNA交联物的分析应确定这一点 REV在该位置终止剪接体组装。

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.