Baker B, Muckenthaler M, Vives E, Blanchard A, Braddock M, Nacken W, Kingsman A J, Kingsman S M
Glaxo Group Research and Development Ltd, Greenford, Middlesex, UK.
Nucleic Acids Res. 1994 Aug 25;22(16):3365-72. doi: 10.1093/nar/22.16.3365.
The Tat protein binds to TAR RNA to stimulate the expression of the human immunodeficiency virus type 1 (HIV-1) genome. Tat is an 86 amino acid protein that contains a short region of basic residues (aa49-aa57) that are required for RNA binding and TAR is a 59 nucleotide stem-loop with a tripyrimidine bulge in the upper stem. TAR is located at the 5' end of all viral RNAs. In vitro, Tat specifically interacts with TAR by recognising the sequence of the bulge and upper stem, with no requirement for the loop. However, in vivo the loop sequence is critical for activation, implying a requirement for accessory cellular TAR RNA binding factors. A number of TAR binding cellular factors have been identified in cell extracts and various models for the function of these factors have been suggested, including roles as coactivators and inhibitors. We have now identified a novel 38 kD cellular factor that has little general, single-stranded or double-stranded RNA binding activity, but that specifically recognises the bulge and upper stem region of TAR. The protein, referred to as BBP (bulge binding protein), is conserved in mammalian and amphibian cells and in Schizosaccharomyces pombe but is not found in Saccharomyces cerevisiae. BBP is an effective competitive inhibitor of Tat binding to TAR in vitro. Our data suggest that the bulge-stem recognition motif in TAR is used to mediate cellular factor/RNA interactions and indicates that Tat action might be inhibited by such competing reactions in vivo.
Tat蛋白与TAR RNA结合，以刺激1型人类免疫缺陷病毒（HIV-1）基因组的表达。Tat是一种由86个氨基酸组成的蛋白质，包含一段短的碱性残基区域（第49至57位氨基酸），这是RNA结合所必需的，而TAR是一个59个核苷酸的茎环结构，在上部茎中有一个三嘧啶凸起。TAR位于所有病毒RNA的5'端。在体外，Tat通过识别凸起和上部茎的序列与TAR特异性相互作用，而对环没有要求。然而，在体内，环序列对于激活至关重要，这意味着需要辅助性细胞TAR RNA结合因子。已经在细胞提取物中鉴定出许多TAR结合细胞因子，并提出了这些因子功能的各种模型，包括作为共激活剂和抑制剂的作用。我们现在鉴定出一种新的38 kD细胞因子，它几乎没有一般的单链或双链RNA结合活性，但能特异性识别TAR的凸起和上部茎区域。这种蛋白质被称为BBP（凸起结合蛋白），在哺乳动物、两栖动物细胞和粟酒裂殖酵母中保守，但在酿酒酵母中未发现。BBP在体外是Tat与TAR结合的有效竞争性抑制剂。我们的数据表明，TAR中的凸起-茎识别基序用于介导细胞因子/RNA相互作用，并表明在体内这种竞争性反应可能会抑制Tat的作用。
