BIOCHEMICAL-CHARACTERIZATION OF BINDING OF MULTIPLE HIV-1 REV MONOMERIC PROTEINS TO THE REV RESPONSIVE ELEMENT
BIOCHEMICAL-CHARACTERIZATION OF BINDING OF MULTIPLE HIV-1 REV MONOMERIC PROTEINS TO THE REV RESPONSIVE ELEMENT
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DOI：
10.1021/bi00090a028
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发表时间：
1993-10-05
期刊：
影响因子：
2.9
通讯作者：
RUSCHE, JR
中科院分区：
文献类型：
--
作者：
DALY, TJ;DOTEN, RC;RUSCHE, JR
重组HIV-1 Rev蛋白通过与β-葡萄糖苷酸酶基因的翻译偶联在大肠杆菌中高效表达，并与Rev反应元件(RRE)具有较高的亲和力和特异性。使用凝胶位移分析观察到复杂的REV依赖的结合模式，在化学计量浓度的RRE存在下，该结合模式可以简化为一条或两条主要条带。这些条带与一系列均聚物RNA物种的竞争表明，REV本质上是一种PolyG结合蛋白，尽管PolyI也被证明竞争特定的RRE结合。用I-125标记的REV测定了依赖于REV的凝胶位移络合物的化学计量比。结果表明，最稳定、最低迁移率的络合物每RRE含有7~8个REV分子，而迁移最快的两个络合物分别具有每个RRE 1个和2个REV分子的比率。利用Hill方程作为协同作用的模型，通过对直接硝化纤维素膜结合实验的拟合，得到了n(App)=2的Hill系数，反映了在平衡结合条件下REV分子协同结合在RRE上的情况。当离子强度从0.0M增加到0.3M时，协同REV与RRE的结合减少，但REV相对于反义RNA的特异性增加了10万倍。当REV与RRE的摩尔比大于2时，REV与RRE解离，t1/2约为20-25min。在摩尔比1以下，关闭速率增加到7到9分钟之间，这表明第二个REV单体的结合足以稳定RRE上的初始结合REV物种。这些数据都支持这样的观点，即虽然REV具有内在的多聚化活性，但它作为单体与RRE相互作用，并通过至少一个额外的REV分子结合稳定在RRE上。
Recombinant HIV-1 Rev protein was overexpressed in Escherichia coli using translational coupling to the beta-glucuronidase gene and demonstrated to interact with high affinity and specificity with the Rev responsive element (RRE). A complex Rev-dependent binding pattern was observed using the gel shift assay which could be simplified to one or two primary bands in the presence of stoichiometric concentrations of RRE. Competition of these bands with a series of homopolymer RNA species demonstrated that Rev is essentially a poly-G binding protein, although poly-I was also shown to compete for specific RRE binding. The stoichiometry of the Rev-dependent gel shift complexes was determined using I-125-labeled Rev. The stable, lowest mobility complex was determined to possess a ratio of between 7 and 8 Rev molecules per RRE containing RNA fragment while the two fastest migrating complexes contained ratios of one and two Rev molecules per RRE, respectively. Using the Hill equation as a model for cooperative interactions, a Hill coefficient of n(app) = 2 was obtained from fitting of direct nitrocellulose filter binding assays, reflecting cooperatively bound Rev molecules on the RRE under equilibrium binding conditions. An increase in ionic strength from 0.0 to 0.3 M NaCl reduced cooperative Rev binding to the RRE, but specificity of Rev for the RRE relative to antisense RNA was increased 100 000-fold. At molar ratios of Rev to RRE above 2, Rev dissociated from the RRE with a T1/2 of approximately 20-25 min. Below a molar ratio of 1, the off-rate increased to between 7 and 9 min, suggesting that binding of a second Rev monomer is sufficient to stabilize the initial bound Rev species on the RRE. The data all support the contention that although Rev possesses an intrinsic multimerization activity, it interacts with the RRE as a monomer and is stabilized on the RRE by binding of at least one additional Rev molecule.