Mundigala Hansini, Michaux Jonathan B, Feig Andrew L, Ennifar Eric, Rueda David
Department of Chemistry, Wayne State University, Detroit, MI 48236, USA.
Department of Chemistry, Wayne State University, Detroit, MI 48236, USA
Nucleic Acids Res. 2014 Jun;42(11):7281-9. doi: 10.1093/nar/gku332. Epub 2014 May 9.
The HIV-1 dimerization initiation sequence (DIS) is a conserved palindrome in the apical loop of a conserved hairpin motif in the 5'-untranslated region of its RNA genome. DIS hairpin plays an important role in genome dimerization by forming a 'kissing complex' between two complementary hairpins. Understanding the kinetics of this interaction is key to exploiting DIS as a possible human immunodeficiency virus (HIV) drug target. Here, we present a single-molecule Förster resonance energy transfer (smFRET) study of the dimerization reaction kinetics. Our data show the real-time formation and dissociation dynamics of individual kissing complexes, as well as the formation of the mature extended duplex complex that is ultimately required for virion packaging. Interestingly, the single-molecule trajectories reveal the presence of a previously unobserved bent intermediate required for extended duplex formation. The universally conserved A272 is essential for the formation of this intermediate, which is stabilized by Mg(2+), but not by K(+) cations. We propose a 3D model of a possible bent intermediate and a minimal dimerization pathway consisting of three steps with two obligatory intermediates (kissing complex and bent intermediate) and driven by Mg(2+) ions.
HIV-1二聚化起始序列（DIS）是其RNA基因组5'非翻译区保守发夹基序顶端环中的一个保守回文序列。DIS发夹通过在两个互补发夹之间形成“亲吻复合物”在基因组二聚化中发挥重要作用。了解这种相互作用的动力学是将DIS开发为可能的人类免疫缺陷病毒（HIV）药物靶点的关键。在此，我们展示了一项关于二聚化反应动力学的单分子荧光共振能量转移（smFRET）研究。我们的数据显示了单个亲吻复合物的实时形成和解离动力学，以及病毒体包装最终所需的成熟延伸双链复合物的形成。有趣的是，单分子轨迹揭示了延伸双链形成所需的一种先前未观察到的弯曲中间体的存在。普遍保守的A272对于该中间体的形成至关重要，该中间体由Mg(2+)稳定，但不由K(+)阳离子稳定。我们提出了一个可能的弯曲中间体的三维模型以及一个由三个步骤组成的最小二聚化途径，该途径有两个必需中间体（亲吻复合物和弯曲中间体）并由Mg(2+)离子驱动。
