Department of Chemistry and Biochemistry, Center for Biomolecular Structure and Organization, University of Maryland, College Park, MD 20742-3360, USA.
J Biomol NMR. 2012 Jan;52(1):65-77. doi: 10.1007/s10858-011-9582-5. Epub 2011 Nov 17.
Selective isotopic labeling provides an unparalleled window within which to study the structure and dynamics of RNAs by high resolution NMR spectroscopy. Unlike commonly used carbon sources, the asymmetry of (13)C-labeled pyruvate provides selective labeling in both the ribose and base moieties of nucleotides using Escherichia coli variants, that until now were not feasible. Here we show that an E. coli mutant strain that lacks succinate and malate dehydrogenases (DL323) and grown on [3-(13)C]-pyruvate affords ribonucleotides with site specific labeling at C5' (95%) and C1' (42%) and minimal enrichment elsewhere in the ribose ring. Enrichment is also achieved at purine C2 and C8 (95%) and pyrimidine C5 (100%) positions with minimal labeling at pyrimidine C6 and purine C5 positions. These labeling patterns contrast with those obtained with DL323 E. coli grown on [1, 3-(13)C]-glycerol for which the ribose ring is labeled in all but the C4' carbon position, leading to multiplet splitting of the C1', C2' and C3' carbon atoms. The usefulness of these labeling patterns is demonstrated with a 27-nt RNA fragment derived from the 30S ribosomal subunit. Removal of the strong magnetic coupling within the ribose and base leads to increased sensitivity, substantial simplification of NMR spectra, and more precise and accurate dynamic parameters derived from NMR relaxation measurements. Thus these new labels offer valuable probes for characterizing the structure and dynamics of RNA that were previously limited by the constraint of uniformly labeled nucleotides.
选择性同位素标记为通过高分辨率 NMR 光谱研究 RNA 的结构和动态提供了无与伦比的窗口。与常用的碳源不同，（13）C 标记的丙酮酸的不对称性为大肠杆菌变体中的核苷酸的核糖和碱基部分提供了选择性标记，直到现在这是不可行的。在这里，我们展示了一种缺乏琥珀酸和苹果酸脱氢酶（DL323）的大肠杆菌突变株，在[3-（13）C]-丙酮酸上生长，可以在 C5'（95%）和 C1'（42%）处提供核糖核苷酸的位置特异性标记，而核糖环的其他部位的丰度很低。嘌呤 C2 和 C8（95%）和嘧啶 C5（100%）位置的丰度也得到了实现，嘧啶 C6 和嘌呤 C5 位置的标记很少。这些标记模式与在[1,3-（13）C]-甘油上生长的 DL323 大肠杆菌获得的标记模式形成对比，其中除了 C4'碳原子位置之外，核糖环都被标记，导致 C1'、C2'和 C3'碳原子的多重分裂。这些标记模式的有用性通过来自 30S 核糖体亚基的 27-nt RNA 片段得到证明。去除核糖和碱基内的强磁耦合导致灵敏度增加，NMR 谱的大幅度简化，以及从 NMR 弛豫测量得出的更精确和准确的动态参数。因此，这些新的标记物为表征 RNA 的结构和动态提供了有价值的探针，这些探针以前受到均一标记核苷酸的限制。
