Site-specific labeling of nucleotides for making RNA for high resolution NMR studies using an E. coli strain disabled in the oxidative pentose phosphate pathway.
Site-specific labeling of nucleotides for making RNA for high resolution NMR studies using an E. coli strain disabled in the oxidative pentose phosphate pathway.
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DOI：
10.1007/s10858-010-9405-0
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发表时间：
2010-05
影响因子：
2.7
通讯作者：
Thakur CS
中科院分区：
文献类型：
--
作者：
Dayie TK;Thakur CS
大肠埃希菌（E.大肠杆菌）是一种多用途的生物体，用于制备用稳定同位素（13 C、15 N和/或2 H）标记的核苷酸，用于结构和分子动力学表征。突变株E.缺乏戊糖磷酸途径酶葡萄糖-6-磷酸脱氢酶（K10-1516）的大肠杆菌菌株在Studier基本培养基中的2- 13 C-甘油和15 N-硫酸铵上能够在用于NMR光谱的位点进行标记。然而，13 C-甲酸钠与生长培养基中的13 C-2-甘油组合将标记添加到新位置。在不存在标记的甲酸盐的情况下，由于1 JC 5C 6标量耦合，嘧啶环的C5和C6位置均标记有最小的多重峰分裂。然而，嘌呤环内的C2/C8位点和核糖环内的C1′/C3′/C5′位置具有减少的标记。添加13 C标记的甲酸盐导致碱基C2/C8和核糖C1′/C3′/C5′位置的标记增加;这些新的特异性标记导致分辨共振的数量增加2 - 3倍。甲酸盐和15 N-硫酸铵的这种使用有望进一步扩展这些替代位点特异性标记的效用，以制备用于下游生物物理应用的标记RNA，例如感兴趣的生物相关RNA的结构、动力学和功能研究。
Escherichia coli (E. coli) is a versatile organism for making nucleotides labeled with stable isotopes (13C, 15N, and/or 2H) for structural and molecular dynamics characterizations. Growth of a mutant E. coli strain deficient in the pentose phosphate pathway enzyme glucose-6-phosphate dehydrogenase (K10-1516) on 2-13C-glycerol and 15N-ammonium sulfate in Studier minimal medium enables labeling at sites useful for NMR spectroscopy. However, 13C-sodium formate combined with 13C-2-glycerol in the growth media adds labels to new positions. In the absence of labeled formate, both C5 and C6 positions of the pyrimidine rings are labeled with minimal multiplet splitting due to 1JC5C6 scalar coupling. However, the C2/C8 sites within purine rings and the C1′/C3′/C5′ positions within the ribose rings have reduced labeling. Addition of 13C-labeled formate leads to increased labeling at the base C2/C8 and the ribose C1′/C3′/C5′ positions; these new specific labels result in two- to three-fold increase in the number of resolved resonances. This use of formate and 15N-ammonium sulfate promises to extend further the utility of these alternate site specific labels to make labeled RNA for downstream biophysical applications such as structural, dynamics and functional studies of interesting biologically relevant RNAs.
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解决 RNA 结构生物学中重大问题的关键标记技术。
影响因子：
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