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首页> 外文期刊>Biochemistry >Nucleic acid sequence discrimination by the HIV-1 nucleocapsid protein NCp7: a fluorescence study.
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Nucleic acid sequence discrimination by the HIV-1 nucleocapsid protein NCp7: a fluorescence study.

机译：HIV-1核衣壳蛋白NCp7对核酸序列的识别：荧光研究。

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摘要

The critical functions of the HIV-1 nucleocapsid protein NCp7 in genomic RNA packaging and reverse transcription, essentially rely on interactions with nucleic acids. A significant progress in the knowledge of these interactions has been recently achieved with the NMR-derived structures of NCp7 derivatives in complex with two short sequences of the HIV-1 psi packaging signal, namely ACGCC and the stem-loop 3 (SL3) motif. To further identify the key nucleotides in the formation of both NCp7-d(ACGCC) and NCp7-SL3 complexes, we quantitatively analyzed by steady-state and time-resolved fluorescence, the interaction of NCp7 with d(ACGCC) and SL3 mutants where each nucleotide in interaction with the protein has been systematically substituted. Moreover, by using several NCp7 derivatives, we investigated the contributions of Phe16, Trp37, and Trp61, and the various NCp7 domains, in the binding process. The binding of NCp7 appeared essentially driven by the interaction of the zinc finger domain and notably Trp37 with a G residue, irrespective of its location in the oligonucleotide. The involvement of Trp37 in the binding process depended on its location in the C-terminal finger motif and the proper folding of this motif. Phe16 in the N-terminal finger motif also strongly contributed to the binding energy, while in contrast, Trp61 in the C-terminal domain only marginally interacted with the oligonucleotides. The stem-loop structure of SL3 stabilized the binding of NCp7 by about -7 kJ/mol (at 0.1 M NaCl) by favoring the electrostatic binding of both N- and C-terminal domains. Finally, we found that NCp7 bound to nucleic acid single-stranded regions with the following preference: X(i)()TGX(j)() > X(i)()GXGX(j)() approximately X(i)()TXGX(j)() > X(i)()GX(j)() X(i)()X(j)(), where X corresponds to either A or C. This implies that recognition of nucleic acids by NCp7 may be achieved by a limited number of sites, and hence, no strong affinities are required in order to get a selective binding.
机译：HIV-1核衣壳蛋白NCp7在基因组RNA包装和逆转录中的关键功能基本上依赖于与核酸的相互作用。这些相互作用的知识方面的重大进展是最近通过NCp7衍生物的NMR衍生结构与HIV-1 psi包装信号的两个短序列（即ACGCC和茎环3（SL3）基序）复合形成的。为了进一步鉴定形成NCp7-d（ACGCC）和NCp7-SL3复合物的关键核苷酸，我们通过稳态和时间分辨荧光定量分析了NCp7与d（ACGCC）和SL3突变体的相互作用，与蛋白质相互作用的核苷酸已被系统取代。此外，通过使用几种NCp7衍生物，我们研究了Phe16，Trp37和Trp61以及各种NCp7域在结合过程中的贡献。不论其在寡核苷酸中的位置如何，NCp7的结合基本上都由锌指结构域，尤其是Trp37与G残基的相互作用驱动。 Trp37在结合过程中的参与取决于其在C端手指基序中的位置以及该基序的正确折叠。 N末端手指基序中的Phe16也强烈地促进了结合能，而相比之下，C末端域中的Trp61仅与寡核苷酸微弱地相互作用。 SL3的茎环结构通过促进N和C端结构域的静电结合，使NCp7的结合稳定在-7 kJ / mol（在0.1 M NaCl时）。最后，我们发现NCp7以以下优先级结合到核酸单链区域：X（i）（）TGX（j）（）> X（i）（）GXGX（j）（）约X（i）（ ）TXGX（j）（）> X（i）（）GX（j）（） X（i）（）X（j）（），其中X对应于A或C.这意味着对核酸的识别NCp7可以通过有限数量的位点获得氨基酸，因此，不需要强亲和力即可获得选择性结合。

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