Asymmetry of (13)C labeled 3-pyruvate affords improved site specific labeling of RNA for NMR spectroscopy.
Asymmetry of (13)C labeled 3-pyruvate affords improved site specific labeling of RNA for NMR spectroscopy.
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(13)C 标记的 3-丙酮酸的不对称性为 NMR 光谱提供了改进的 RNA 位点特异性标记。
DOI：
10.1007/s10858-011-9581-6
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发表时间：
2011
影响因子：
2.7
通讯作者：
Dayie,TKwaku
中科院分区：
文献类型：
--
作者：
Thakur,ChandarS;Dayie,TKwaku
选择性同位素标记提供了一个无与伦比的窗口，在其中研究高分辨率核磁共振波谱rna的结构和动力学。与常用的碳源不同，13c标记的丙酮酸的不对称性为使用inge的核苷酸的核糖和碱基部分提供了选择性标记。大肠杆菌，直到现在还不可行。这里我们展示了anE。缺乏琥珀酸脱氢酶和苹果酸脱氢酶（DL323）并生长在[3-13C]-丙酮酸上的共配菌株提供具有C5 ‘（~95%）和C1 ’（~42%）特异位点标记的核糖核苷酸，并且在核糖环的其他地方富集最少。嘌呤C2和C8（~95%）和嘧啶C5（~100%）位置也实现了富集，嘧啶C6和嘌呤C5位置的标记最少。这些标记模式与DL323E的结果相反。在[1,3 - 13c]-甘油上生长，其核糖环在除C4 ‘碳位置外的所有碳位置上都被标记，导致C1 ’， C2 ‘和C3 ’碳原子的多重分裂。从30S核糖体亚基衍生的27-nt RNA片段证明了这些标记模式的有效性。去除核糖和碱基之间的强磁耦合，可以提高灵敏度，大大简化核磁共振光谱，并从核磁共振弛豫测量中获得更精确和准确的动态参数。因此，这些新的标记为表征RNA的结构和动力学提供了有价值的探针，这些探针以前受到统一标记核苷酸的限制。
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 of13C-labeled pyruvate provides selective labeling in both the ribose and base moieties of nucleotides usingE.colivariants, that until now were not feasible. Here we show that anE.colimutant strain that lacks succinate and malate dehydrogenases (DL323) and grown on [3-13C]-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 DL323E.coligrown on [1, 3-13C]-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.