THE CELLULAR FACTOR TRP-185 REGULATES RNA-POLYMERASE-II BINDING TO HIV-1 TAR RNA
THE CELLULAR FACTOR TRP-185 REGULATES RNA-POLYMERASE-II BINDING TO HIV-1 TAR RNA
复制标题
DOI：
10.1002/j.1460-2075.1995.tb00288.x
复制
发表时间：
1995-12-01
期刊：
影响因子：
11.4
通讯作者：
GAYNOR, RB
中科院分区：
文献类型：
--
作者：
WUBAER, F;LANE, WS;GAYNOR, RB
反式激活因子 Tat 对 HIV-1 基因表达的激活依赖于位于转录起始位点（称为 TAR）下游的 RNA 调节元件。为了表征与 TAR RNA 结合并参与 HIV-1 转录调节的细胞因子，对 HeLa 核提取物进行分级并进行 RNA 凝胶延迟分析。该分析表明，只有两种细胞因子，即 RNA 聚合酶 II 和先前表征的 TAR RNA 环结合蛋白 TRP-185，能够与 TAR RNA 特异性结合。为了阐明TRP-185的功能，从HeLa核提取物中纯化了TRP-185，进行了氨基酸微序列分析，并分离了编码TRP-185的cDNA，TRP-185是一种含有1621个氨基酸的新型蛋白质，含有亮氨酸拉链，并且可能是一种新型RNA结合蛾。在凝胶延迟测定中，重组 TRP-185 和 RNA 聚合酶 II 的结合取决于另一组称为细胞辅因子的蛋白质的存在。 TAR RNA 环和突出序列对于 RNA 聚合酶 LI 结合至关重要，而 TRP-185 结合仅依赖于 TAR RNA 环序列。由于发现 TRP-185 和 RNA 聚合酶 II 与 TAR RNA 的结合是相互排斥的，因此我们的结果表明，TRP-185 可以单独发挥作用，也可以与 Tat 联合发挥作用，以脱离 RNA 聚合酶 II，而 RNA 聚合酶 II 在转录延伸过程中与新生合成的 TAR RNA 结合时会停止。
Activation of HIV-1 gene expression by the transactivator Tat is dependent on an RNA regulatory element located downstream of the transcription initiation site known as TAR. To characterize cellular factors that bind to TAR RNA and are involved in the regulation of HIV-1 transcription, HeLa nuclear extract was fractionated and RNA gel-retardation analysis was performed. This analysis indicated that only two cellular factors, RNA polymerase II and the previously characterized TAR RNA loop binding protein TRP-185, were capable of binding specifically to TAR RNA. To elucidate the function of TRP-185, it was purified from HeLa nuclear extract, amino acid microsequence analysis was performed and a cDNA encoding TRP-185 was isolated, TRP-185 is a novel protein of 1621 amino acids which contains a leucine zipper and potentially a novel RNA binding moth. In gel-retardation assays, the binding of both recombinant TRP-185 and RNA polymerase II was dependent on the presence of an additional group of proteins designated cellular cofactors. Both the TAR RNA loop and bulge sequences were critical for RNA polymerase LI binding, while TRP-185 binding was dependent only on TAR RNA loop sequences. Since binding of TRP-185 and RNA polymerase II to TAR RNA was found to be mutually exclusive, our results suggest that TRP-185 may function either alone or in conjunction with Tat to disengage RNA polymerase II which is stalled upon binding to nascently synthesized TAR RNA during transcriptional elongation.