Dingwall C, Ernberg I, Gait M J, Green S M, Heaphy S, Karn J, Lowe A D, Singh M, Skinner M A
Medical Research Council, Laboratory of Molecular Biology, Cambridge, UK.
EMBO J. 1990 Dec;9(12):4145-53. doi: 10.1002/j.1460-2075.1990.tb07637.x.
The HIV-1 trans-activator protein, tat, is an RNA binding protein with a high affinity for a U-rich bulge near the tip of the stem in the RNA stem-loop structure encoded by the trans-activation responsive region (TAR). A Scatchard analysis of tat binding has shown that the purified protein forms a one-to-one complex with HIV-1 TAR RNA with a dissociation constant of Kd = 12 nM. Deletion of the uridine residues in the bulge or substitution with guanine residues produced RNAs with a 6- to 8-fold lower affinity than wild-type TAR. Introduction of a point mutation expected to destabilize base pairing in nearby residues of the TAR stem-loop structure reduced tat binding 10-fold. In contrast, mutations that alter the sequence of the six nucleotide long loop at the tip of TAR RNA structure, and mutations which alter the sequence of the stem whilst preserving Watson-Crick base pairing, do not affect tat binding significantly. There is a direct correlation between the ability of tat to bind to TAR RNA and to activate HIV transcription. Viral LTRs carrying TAR sequences encoding any of the mutations known to produce transcripts which bind tat weakly, are not stimulated efficiently by tat in vivo.
HIV-1反式激活蛋白tat是一种RNA结合蛋白，对反式激活应答区域（TAR）编码的RNA茎环结构茎部顶端富含尿苷的凸起具有高亲和力。对tat结合进行的Scatchard分析表明，纯化后的蛋白与HIV-1 TAR RNA形成一对一复合物，解离常数Kd = 12 nM。凸起处尿苷残基的缺失或被鸟嘌呤残基取代产生的RNA，其亲和力比野生型TAR低6至8倍。预期会破坏TAR茎环结构附近残基碱基配对的点突变使tat结合减少了10倍。相反，改变TAR RNA结构顶端六核苷酸长环序列的突变，以及改变茎部序列同时保留沃森-克里克碱基配对的突变，对tat结合没有显著影响。tat与TAR RNA结合的能力和激活HIV转录的能力之间存在直接相关性。携带编码已知会产生与tat弱结合转录本的任何突变的TAR序列的病毒长末端重复序列，在体内不会被tat有效刺激。
