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首页> 外文期刊>Nucleic Acids Research >Assignment of the L30-mRNA complex using selective isotopic labeling and RNA mutants.
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Assignment of the L30-mRNA complex using selective isotopic labeling and RNA mutants.

机译：使用选择性同位素标记和RNA突变体分配L30-mRNA复合物。

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摘要

The helix-loop-helix structure formed in the pre-mRNA and the mRNA of L30, a ribosomal protein from the yeast Saccharomyces cerevisiae, serves as an auto-regulatory binding site for the protein to suppress the L30 synthesis upon overproduction. Using a 33-nucleotide model RNA, the structures of the L30 binding site RNA in the presence and absence of the protein were investigated using nuclear magnetic resonance (NMR) spectroscopy. Homonuclear and(13)C/(15)N-based resonance assignments and spectral comparisons indicated that the purine-rich internal loop is dynamic in the free RNA but becomes ordered in the presence of L30 protein. Although the resonances in the loop region are sharper and more disperse in the bound RNA, their assignment was extremely challenging, due to spectral complexity and broadened resonances caused by local dynamics. Two strategies, namely selective(13)C/(15)N-labeling and NMR analyses of five complexes with RNA mutants, were used to overcome these difficulties. Only using these approaches could assignment of the internal loop resonances and identification of the unusual NOEs and nucleotide conformations within the internal loop be made. In the case of structural determination of the L30-mRNA complex, it was critical to be able to take advantage of the available biochemical information in order to complete the structure determination.
机译：在pre-mRNA中形成的螺旋-环-螺旋结构和L30（一种来自酿酒酵母）的核糖体蛋白的mRNA作为该蛋白的自动调节结合位点，可抑制过量生产时L30的合成。使用33个核苷酸的模型RNA，使用核磁共振（NMR）光谱研究存在和不存在蛋白质时L30结合位点RNA的结构。基于Homonuclear和（13）C /（15）N的共振分配和光谱比较表明，富含嘌呤的内部环在自由RNA中是动态的，但在L30蛋白存在下变得有序。尽管在环区域中的共振更加尖锐，并且在结合的RNA中更分散，但是由于光谱复杂性和局部动力学导致的共振变宽，它们的分配非常具有挑战性。两种策略，即选择性（13）C /（15）N-标记和具有RNA突变体的五个复合物的NMR分析，被用来克服这些困难。只有使用这些方法，才能进行内部环共振的分配以及内部环内异常NOE和核苷酸构象的鉴定。对于L30-mRNA复合物的结构测定，至关重要的是能够利用可用的生化信息来完成结构测定。

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