Paoletti Andrew C, Shubsda Michael F, Hudson Bruce S, Borer Philip N
Department of Chemistry, Graduate Program in Structural Biology, Biochemistry, and Biophysics, Syracuse University, Syracuse, New York 13244-4100, USA.
Biochemistry. 2002 Dec 24;41(51):15423-8. doi: 10.1021/bi026307n.
Efficient packaging of genomic RNA into new HIV-1 virus particles requires that nucleocapsid domains of precursor proteins bind the SL3 tetraloop (G317-G-A-G320) from the 5'-untranslated region. This paper presents the affinities of 35 RNA variants of SL3 for the mature 55mer NC protein, as measured by fluorescence quenching of tryptophan-37 in the protein by nucleobases. The 1:1 complexes that form in 0.2 M NaCl have dissociation constants ranging from 8 nM (GGUG) to 20 microM (GAUA). The highly conserved (GGAG) sequence for the wild type is not the most stable (K(d) = 28 nM), suggesting that other selective pressures beyond the stability of the complex must be satisfied. The leading requirement for strong interaction is for G320, followed closely by G318. Replacing either with U, A, or C reduces affinity by a factor of 15-120. NC-domains from multiple proteins combine to recognize unpaired G(2)-loci, where two guanines are in close proximity. We have previously measured affinities of the NC protein for the important stem-loops of the major packaging domain [Shubsda, M. F., Paoletti, A. C., Hudson, B. S., and Borer, P. N. (2002) Biochemistry 41, 5276-82]. Comparison with the present work shows that the nature of the stem also modulates NC-RNA interactions. Placing the G(2)-loci from the apical SL2 or SL1 loops on the SL3 stem increases affinity by a factor of 2-3, while placing the SL4 loop on the SL3 stem reduces affinity 50-fold. These results are interesting in the context of RNA-protein interaction, as well as for the discovery of antiNC agents for AIDS therapy.
将基因组RNA高效包装到新的HIV-1病毒颗粒中，需要前体蛋白的核衣壳结构域与5'-非翻译区的SL3四环（G317-G-A-G320）结合。本文介绍了通过核碱基对蛋白质中色氨酸-37的荧光猝灭测量的35种SL3 RNA变体与成熟的55聚体NC蛋白的亲和力。在0.2 M NaCl中形成的1:1复合物的解离常数范围为8 nM（GGUG）至20 μM（GAUA）。野生型的高度保守序列（GGAG）并非最稳定（K(d)=28 nM），这表明除了复合物的稳定性之外，还必须满足其他选择压力。强相互作用的主要要求是G320，其次是G318。用U、A或C取代其中任何一个都会使亲和力降低15至120倍。来自多种蛋白质的NC结构域结合起来识别未配对的G(2)位点，其中两个鸟嘌呤紧密相邻。我们之前已经测量了NC蛋白对主要包装结构域重要茎环的亲和力[Shubsda, M. F., Paoletti, A. C., Hudson, B. S., and Borer, P. N. (2002) Biochemistry 41, 5276-82]。与当前工作的比较表明，茎的性质也会调节NC-RNA相互作用。将来自顶端SL2或SL1环的G(2)位点置于SL3茎上会使亲和力提高2至3倍，而将SL4环置于SL3茎上会使亲和力降低50倍。这些结果在RNA-蛋白质相互作用的背景下以及在发现用于艾滋病治疗的抗NC药物方面都很有趣。
