De Tapia M, Metzler V, Mougel M, Ehresmann B, Ehresmann C
Unité Propre de Recherche 9002 du CNRS, Institut de Biologie Moléculaire et Cellulaire, Strasbourg, France.
Biochemistry. 1998 Apr 28;37(17):6077-85. doi: 10.1021/bi9800303.
Genomic RNAs from retroviruses are packaged as dimers of two identical RNA molecules. In Moloney murine leukemia virus, a stem-loop structure (H1) located in the encapsidation domain Psi (nucleotides 215-564) was postulated to trigger RNA dimerization through base pairing between auto complementary sequences. The Psi domain also contains two other stem-loop structures (H2 and H3) that are essential for RNA packaging. Since it was suspected than H1 is not the only element involved in RNA dimerization, we systematically investigated the dimerization capacity of several subdomains of the first 725 nucleotides of genomic RNA. The efficiency of dimerization of the various RNAs was estimated by measuring their apparent dissociation constants, and the specificity was tested by competition experiments. Our results indicate that the specificity of dimerization of RNA nucleotides 1-725 is driven by motifs H1-H3 in domain Psi. To define the relative contributions of these elements, RNA deletion mutants containing different combinations of H1-H3 were constructed and further analyzed in competition and kinetic experiments. Our results confirm the importance of H1 in triggering dimerization and shed new light on the mechanism of dimerization. H1 is required to provide a stable dimer, probably through the formation of extended intermolecular interactions. However, H1-mediated association is a slow process that is kinetically enhanced by H3, and to a lesser extent by H2. We suggest that they facilitate the recognition between the two RNAs, most likely through their conserved GACG loops. Our results reinforce the idea that dimerization and packaging are two closely related processes.
逆转录病毒的基因组RNA以两个相同RNA分子的二聚体形式被包装。在莫洛尼鼠白血病病毒中，位于包装结构域Ψ（核苷酸215 - 564）中的一个茎环结构（H1）被推测通过自身互补序列之间的碱基配对来触发RNA二聚化。Ψ结构域还包含另外两个对RNA包装至关重要的茎环结构（H2和H3）。由于怀疑H1不是参与RNA二聚化的唯一元件，我们系统地研究了基因组RNA前725个核苷酸的几个亚结构域的二聚化能力。通过测量各种RNA的表观解离常数来估计其二聚化效率，并通过竞争实验测试其特异性。我们的结果表明，RNA核苷酸1 - 725的二聚化特异性是由Ψ结构域中的基序H1 - H3驱动的。为了确定这些元件的相对贡献，构建了包含H1 - H3不同组合的RNA缺失突变体，并在竞争和动力学实验中进一步分析。我们的结果证实了H1在触发二聚化中的重要性，并为二聚化机制提供了新的线索。H1可能通过形成扩展的分子间相互作用来提供稳定的二聚体。然而，H1介导的缔合是一个缓慢的过程，在动力学上由H3增强，在较小程度上由H2增强。我们认为它们促进了两个RNA之间的识别，很可能是通过它们保守的GACG环。我们的结果强化了二聚化和包装是两个密切相关过程的观点。
