Biomass production of site selective 13C/15N nucleotides using wild type and a transketolase E. coli mutant for labeling RNA for high resolution NMR.
Biomass production of site selective 13C/15N nucleotides using wild type and a transketolase E. coli mutant for labeling RNA for high resolution NMR.
复制标题
使用野生型和转酮酶大肠杆菌突变体生产位点选择性 13C/15N 核苷酸，用于标记 RNA 以进行高分辨率 NMR。
DOI：
10.1007/s10858-011-9586-1
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发表时间：
2012
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;Luo,Yiling;Chen,Bin;Eldho,NadukkudyV;Dayie,TKwaku
通过NMR表征核酸的结构和动力学显著受益于位置特异性标记的核苷酸。这里有一个。转酮醇酶基因（tktA）缺陷并在不同碳位点标记的葡萄糖上生长的大肠杆菌菌株显示出有利于有用核苷酸的成本有效和大规模生产。这些核苷酸在C1′和C5′中进行位点特异性标记，核糖环内的干扰最小。为了证明这种标记方法的实用性，新的位点特异性标记和均匀标记的核苷酸被用来合成含有褐藻组II内含子自剪接核酶的催化必需结构域5（D5）的36-nt RNA。D5 RNA用于通过NMR光谱探测的结合和弛豫研究。D5 RNA中涉及结合Mg 2+离子的关键核苷酸得到很好的解析。因此，与使用均匀标记的核苷酸获得的光谱相比，使用选择性标记的核苷酸获得的光谱具有更高的信噪比。因此，与均匀13 C/15 N标记的核苷酸相比，这些特异性标记的核苷酸消除了含氮碱基和核糖环内的广泛13 C-13 C偶联，产生更少拥挤和更高分辨的NMR光谱，以及准确的弛豫速率，而不需要恒定时间或带选择性去耦NMR实验。因此，这些位置选择性标记的核苷酸应该在生物学上感兴趣的RNA分子的NMR分析中得到广泛应用。
Characterization of the structure and dynamics of nucleic acids by NMR benefits significantly from position specifically labeled nucleotides. Here anE. colistrain deficient in the transketolase gene (tktA) and grown on glucose that is labeled at different carbon sites is shown to facilitate cost-effective and large scale production of useful nucleotides. These nucleotides are site specifically labeled in C1′ and C5′ with minimal scrambling within the ribose ring. To demonstrate the utility of this labeling approach, the new site-specific labeled and the uniformly labeled nucleotides were used to synthesize a 36-nt RNA containing the catalytically essential domain 5 (D5) of the brown algae group II intron self-splicing ribozyme. The D5 RNA was used in binding and relaxation studies probed by NMR spectroscopy. Key nucleotides in the D5 RNA that are implicated in binding Mg2+ions are well resolved. As a result, spectra obtained using selectively labeled nucleotides have higher signal-to-noise ratio compared to those obtained using uniformly labeled nucleotides. Thus, compared to the uniformly13C/15N-labeled nucleotides, these specifically labeled nucleotides eliminate the extensive13C–13C coupling within the nitrogenous base and ribose ring, give rise to less crowded and more resolved NMR spectra, and accurate relaxation rates without the need for constant-time or band-selective decoupled NMR experiments. These position selective labeled nucleotides should, therefore, find wide use in NMR analysis of biologically interesting RNA molecules.