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首页> 外文期刊>Biochemistry >Monomer-dimer equilibrium constants of RNA in the dimer initiation site of human immunodeficiency virus type 1.
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Monomer-dimer equilibrium constants of RNA in the dimer initiation site of human immunodeficiency virus type 1.

机译：1型人类免疫缺陷病毒二聚体起始位点中RNA的二聚体-二聚体平衡常数。

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The genome of the human immunodeficiency virus (HIV) exists as a dimer of two identical RNA molecules hydrogen bonded to each other near their 5' ends. The dimer, known to be important for viral infectivity, is formed by two monomers interacting through a stem-loop structure called the dimer initiation site (DIS). An initially formed intermediate, the "kissing" dimer, is unstable and rearranges to the stable, duplex form. In this report we use nondenaturing polyacrylamide gel electrophoresis to measure the monomer-dimer equilibrium constant of three RNA sequences, 41-, 27-, and 19-mers, located in the DIS of the MAL isolate of HIV-1. Experiments in which the RNA was equilibrated at various temperatures before electrophoresis revealed that interconversion is rapid for all the sequences, so that they reach equilibrium in the loading well of the gel at 5 degrees C before they enter the gel proper. However, interconversion kinetics in the gel are slow, so autoradiographic spot intensities can be used to measure the amounts of monomer and dimer present when the sample entered the gel. After correction for the amount of RNA added with the radiolabel and dilution of samples in the loading well of the gel, dimerization equilibrium constants were calculated from spot intensities. The calculated values of the dimerization constant K at 5 degrees C were approximately 10(5), approximately 10(6), and approximately 10(8) M(-1) for the 41-, 27-, and 19-mers, respectively, in solutions of ionic strength, I, of about 100 mM. The decrease in K by three orders of magnitude between the 19-mer and 41-mer is due in part to the change in rotational entropy of rodlike molecules on dimerization and in part to the increased conformational entropy of the monomers. As expected, increased ionic strength increases the dimerization constant for all three RNAs. For the 41-mer, however, K has a maximum value at I approximately 140 mM. The origin of the decrease in K for higher I is unknown but it may be due to formation of species (perhaps higher order oligomers) that do not enter the gel. The 41-mer exists in two dimeric forms assigned to the kissing and duplex dimers. The ratio of kissing to duplex form at 5 degrees C is 0.48 +/- 0.22 at I = 113 mM and 0.91 +/- 0.35 at I = 183 mM. The observed decrease in K with RNA length suggests that the dimerization constant of the packaging region of HIV-1 is small, < approximately 10(5) M(-1), implying that the nucleocapsid protein is important in promoting dimerization in the capsid of the virus.
机译：人类免疫缺陷病毒（HIV）的基因组以两个相同的氢分子在5'端附近相互氢键结合的二聚体形式存在。已知对病毒感染性重要的二聚体是由两个单体通过称为二聚体起始位点（DIS）的茎环结构相互作用而形成的。最初形成的中间体“接吻”二聚体不稳定，并重新排列成稳定的双链形式。在本报告中，我们使用非变性聚丙烯酰胺凝胶电泳来测量位于HIV-1 MAL分离物中DIS的三个RNA序列（41、27和19聚体）的单体二聚体平衡常数。在电泳前在各种温度下平衡RNA的实验表明，所有序列的相互转换都很迅速，因此它们在进入凝胶之前，在5摄氏度的凝胶加载孔中达到平衡。但是，凝胶中的相互转化动力学很慢，因此可以使用放射自显影点强度来测量样品进入凝胶时存在的单体和二聚体的量。校正添加有放射性标记的RNA的量并在凝胶上样孔中稀释样品后，从光点强度计算出二聚化平衡常数。对于41聚体，27聚体和19聚体，在5摄氏度时二聚常数K的计算值分别约为10（5），约10（6）和约10（8）M（-1）。在离子强度I的溶液中约为100 mM。在19-mer和41-mer之间K降低了三个数量级，部分是由于二聚作用时棒状分子旋转熵的变化，部分是由于单体构象熵的增加。如预期的那样，增加的离子强度会增加所有三个RNA的二聚化常数。然而，对于41聚体，K在I处具有最大值，约为140mM。对于较高的I，K降低的原因尚不清楚，但这可能是由于未进入凝胶的物质（也许是高阶低聚物）的形成所致。 41聚体以两种二聚体形式存在，分别分配给接吻和二聚体二聚体。在5摄氏度时，I = 113 mM时亲吻形式与双链体形式之比为0.48 +/- 0.22，在I = 183 mM处为0.91 +/- 0.35。观察到的K随RNA长度的减少表明HIV-1包装区域的二聚常数很小，<10（5）M（-1），这表明核衣壳蛋白在促进壳衣壳的二聚作用中很重要。病毒。

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