Mély Y, de Rocquigny H, Sorinas-Jimeno M, Keith G, Roques B P, Marquet R, Gérard D
Laboratoire de Biophysique, URA 491 du CNRS, Université Louis Pasteur de Strasbourg I, Faculté de Pharmacie, Illkirch, France.
J Biol Chem. 1995 Jan 27;270(4):1650-6. doi: 10.1074/jbc.270.4.1650.
The nucleocapsid protein NCp7 of human immunodeficiency virus, type 1, is a key component in the viral life cycle. Since, the first common step of all reported NCp7 activities corresponds to a nucleic acid-binding step, the NCp7 binding parameters to the natural primer tRNA(3Lys) were investigated. Using NCp7 intrinsic fluorescence, we found that (i) in 0.1 M NaCl, NCp7 bound noncooperatively to tRNA(3Lys) with a Kobs = 3.2 x 10(6) M-1 association constant and a n = 6 binding site size, (ii) four ionic interactions were formed in the NCp7.tRNA(3Lys) complex, and (iii) nonelectrostatic factors provided about 60% of the binding energy. These binding parameters were not significantly altered when the natural tRNA(3Lys) was replaced by either an in vitro synthetic tRNA(3Lys) transcript, the heterologous yeast tRNA(Phe) or the structurally unrelated 5 S RNA from Escherichia coli. Moreover, the environment of the intrinsic fluorescent reporters (Trp37 and Trp61) was similar in the various complexes. Finally, experiments performed at low protein concentration provide no evidence of high affinity binding sites. Taken together, our data strongly suggested an essentially nonspecific binding of NCp7 to tRNA(3Lys) and thus did not seem to support a direct role of NCp7, per se, in the selection of tRNA(3Lys) from the pool of cellular tRNAs.
人类免疫缺陷病毒1型的核衣壳蛋白NCp7是病毒生命周期中的关键成分。由于所有已报道的NCp7活性的首个共同步骤都对应于核酸结合步骤，因此对NCp7与天然引物tRNA（3Lys）的结合参数进行了研究。利用NCp7的固有荧光，我们发现：（i）在0.1 M NaCl中，NCp7以非协同方式结合tRNA（3Lys），其观测结合常数Kobs = 3.2 x 10（6）M-1，结合位点大小n = 6；（ii）在NCp7.tRNA（3Lys）复合物中形成了四个离子相互作用；（iii）非静电因素提供了约60%的结合能。当天然tRNA（3Lys）被体外合成的tRNA（3Lys）转录本、异源酵母tRNA（Phe）或来自大肠杆菌的结构不相关的5 S RNA取代时，这些结合参数没有显著改变。此外，在各种复合物中，固有荧光报告基团（Trp37和Trp61）的环境相似。最后，在低蛋白浓度下进行的实验没有提供高亲和力结合位点的证据。综上所述，我们的数据强烈表明NCp7与tRNA（3Lys）的结合本质上是非特异性的，因此似乎不支持NCp7本身在从细胞tRNA池中选择tRNA（3Lys）方面的直接作用。
