Fong S E, Smanik P, Thais T, Smith M C, Jaskunas S R
Lilly Research Laboratories, Lilly Corporate Center, Indianapolis, IN 46285-0438, USA.
J Virol Methods. 1997 Jun;66(1):91-101. doi: 10.1016/s0166-0934(97)00039-6.
Gene expression from the human immunodeficiency virus 1 (HIV-1) is greatly enhanced by binding of the virally encoded Tat protein to a 59-base RNA stem-loop structure, the Transactivation Responsive Element (TAR), located at the 5'-termini of all viral transcripts. This interaction was investigated in vitro using 32P-labelled TAR and highly purified Tat in which cysteine residues were blocked by sulpitolysis (S-Tat). It is shown that specific complex formation between S-Tat and TAR can occur in the presence of urea, with urea concentrations between 5 and 6 M causing an approximately two-fold increase in the level of binding. Two conditions favoring RNA secondary structure, low temperature (0 degree C) and the presence of divalent cations (Mg2+), diminished the level of specific binding. These observations suggest that the presence of mild denaturants promoted macromolecular refolding or rearrangement in a manner that increased the number of molecules available for binding, and present a general method for studying protein/RNA interactions where analysis has been obstructed by improper protein or RNA conformation.
病毒编码的Tat蛋白与位于所有病毒转录本5'末端的59个碱基的RNA茎环结构（反式激活应答元件，TAR）结合，可极大增强人类免疫缺陷病毒1型（HIV-1）的基因表达。利用32P标记的TAR和经亚硫酸解阻断半胱氨酸残基的高度纯化的Tat（S-Tat）在体外研究了这种相互作用。结果表明，在存在尿素的情况下，S-Tat与TAR之间可形成特异性复合物，尿素浓度在5至6 M之间时，结合水平大约增加两倍。有利于RNA二级结构的两个条件，即低温（0℃）和二价阳离子（Mg2+）的存在，降低了特异性结合水平。这些观察结果表明，温和变性剂的存在以增加可用于结合的分子数量的方式促进了大分子的重折叠或重排，并提出了一种研究蛋白质/RNA相互作用的通用方法，其中分析因蛋白质或RNA构象不当而受阻。
