Stem-loop structure in the 5' region of potato virus X genome required for plus-strand RNA accumulation.
Stem-loop structure in the 5' region of potato virus X genome required for plus-strand RNA accumulation.
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马铃薯病毒 X 基因组 5 区域的茎环结构是正链 RNA 积累所需的。
DOI：
10.1006/jmbi.1998.2174
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发表时间：
1998
期刊：
影响因子：
--
通讯作者：
Hemenway,C
中科院分区：
文献类型：
--
作者：
Miller,ED;Plante,CA;Kim,KH;Brown,JW;Hemenway,C
计算机生成的热力学预测和溶液结构探测表明，在马铃薯X病毒（PVX）RNA的5′ 230 nt内存在两种茎环结构，茎环1（SL 1; nt 32-106）和茎环2（SL 2; nt 143-183）。由于SL 1的存在进一步支持几个PVX菌株的共变异分析，这种结构的功能意义进行了研究，在烟草原生质体系统中的定点突变分析。一般来说，减少基因组正链RNA积累的突变同样影响外壳蛋白的积累，表明亚基因组正链RNA也受到影响。相反，负链RNA水平保持相对不变。SL 1茎C（SC）区的突变分析表明，配对比序列更重要，这与协变异分析一致。增加SC或茎D（SD）的长度和稳定性的改变对正链RNA积累是有害的。SC和SD之间的内环C的形成以及该环内的特定核苷酸也是必需的。对末端GAAA四环进行了几处修饰，这是一种已知的增强RNA稳定性的基序。GANA和GAAG基序均导致野生型水平的RNA积累。然而，UUCG四环是有害的，表明该元件的序列不仅提供结构的稳定性，而且很重要。这些数据表明，SL 1的多种特征对于PVX正链RNA的积累至关重要。
Computer-generated thermodynamic predictions and solution structure probing indicated two stem-loop structures, stem-loop 1 (SL1; nt 32-106) and stem-loop 2 (SL2; nt 143-183), within the 5′ 230 nt of potato virus X (PVX) RNA. Because the existence of SL1 was further supported by covariation analysis of several PVX strains, the functional significance of this structure was investigated by site-directed mutational analysis in a tobacco protoplast system. In general, mutations that reduced genomic plus-strand RNA accumulation similarly affected coat protein accumulation, indicating that subgenomic plus-strand RNA was also affected. In contrast, minus-strand RNA levels remained relatively unchanged. Mutational analysis of the stem C (SC) region of SL1 indicated that pairing was more important than sequence, which was consistent with the covariation analysis. Alterations that increased length and stability of either SC or stem D (SD) were deleterious to plus-strand RNA accumulation. The formation of internal loop C between SC and SD, as well as specific nucleotides within this loop, were also required. Several modifications were made to the terminal GAAA tetraloop, a motif known for enhanced RNA stability. Both GANA and GAAG motifs resulted in wild-type levels of RNA accumulation. However, a UUCG tetraloop was detrimental, indicating that the sequence of this element was important beyond just providing stabilization of the structure. These data indicate that multiple features of SL1 are critical for accumulation of PVX plus-strand RNA.
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