Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, Moscow 119991, Russia.
J Biomol Struct Dyn. 2011 Jun;28(6):939-47. doi: 10.1080/07391102.2011.10508620.
Viral maturation of HIV-1 involves refolding of its genomic RNA, which is believed to include a rearrangement of the SL1 stem-loop from a metastable conformation called kissing loop dimer (KD) to a stable one termed extended dimer (ED). To investigate this rearrangement in vitro we have studied the thermal melting of the RNA dimers formed by slightly modified 23-nucleotide SL1 RNA of HIV-1 Mal. Local structural changes in the RNA dimers during the melting were monitored by changes in the fluorescence of 2-aminopurine (2AP) incorporated in predetermined positions of RNA. We have shown that the stem regions of both preformed KD and ED melt in the temperature interval from 75 ° C to 90 ° C. Kissing loop interface of the KD RNA is found to be disrupted at lower temperatures from 20 ° C to 55 ° C, at which the stem regions remain intact. Conversion of the preformed KD to ED overcoming the kinetic barrier occurs between 55 ° C and 65 ° C. The melting of "loop-loop" regions in both preformed and newly formed EDs takes place around 70 ° C. Our finding that thermoinduced KD-to-ED conversion is preceded by transient dissociation of loop-loop interface disagrees with a common idea of strand exchange without disruption of loop-loop-contact.
HIV-1 的病毒成熟涉及基因组 RNA 的重折叠，据信这包括 SL1 发夹环从一种称为亲吻环二聚体 (KD) 的亚稳态构象到一种称为扩展二聚体 (ED) 的稳定构象的重排。为了在体外研究这种重排，我们研究了略微修饰的 HIV-1 Mal 的 23 个核苷酸 SL1 RNA 形成的 RNA 二聚体的热融解。通过在 RNA 的预定位置掺入的 2-氨基嘌呤 (2AP) 的荧光变化来监测 RNA 二聚体在融解过程中的局部结构变化。我们已经表明，预形成的 KD 和 ED 的茎区在从 75°C 到 90°C 的温度范围内融解。在从 20°C 到 55°C 的较低温度下，KD RNA 的亲吻环界面被发现被破坏，而茎区保持完整。预形成的 KD 转化为 ED 以克服动力学障碍发生在 55°C 到 65°C 之间。在预形成和新形成的 ED 中，“环-环”区域的融解发生在大约 70°C。我们的发现表明，热诱导的 KD-ED 转化是由环-环界面的瞬时解离引起的，这与没有破坏环-环接触的链交换的常见观点不一致。
