Mao H, Williamson J R
Department of Chemistry, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
Nucleic Acids Res. 1999 Oct 15;27(20):4059-70. doi: 10.1093/nar/27.20.4059.
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.
酿酒酵母核糖体蛋白L30的前体mRNA和mRNA中形成的螺旋-环-螺旋结构，作为该蛋白的自调控结合位点，在过量产生时抑制L30的合成。使用一个33个核苷酸的模型RNA，利用核磁共振（NMR）光谱研究了有无该蛋白时L30结合位点RNA的结构。基于同核和（13）C/（15）N的共振归属及光谱比较表明，富含嘌呤的内环在游离RNA中是动态的，但在L30蛋白存在时会变得有序。尽管环区的共振在结合的RNA中更尖锐且更分散，但由于光谱复杂性以及局部动力学导致的共振展宽，其归属极具挑战性。采用了两种策略，即选择性（13）C/（15）N标记和对五个RNA突变体复合物进行NMR分析，以克服这些困难。只有使用这些方法，才能对内环共振进行归属，并识别内环内异常的核Overhauser效应（NOE）和核苷酸构象。在L30-mRNA复合物的结构测定中，能够利用现有的生化信息对于完成结构测定至关重要。
