Fluorine-19 nuclear magnetic resonance studies of the structure of 5-fluorouracil-substituted Escherichia coli transfer RNA.
Fluorine-19 nuclear magnetic resonance studies of the structure of 5-fluorouracil-substituted Escherichia coli transfer RNA.
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5-氟尿嘧啶取代的大肠杆菌转移 RNA 结构的氟 19 核磁共振研究。
DOI：
10.1021/bi00367a053
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发表时间：
1986
期刊：
影响因子：
2.9
通讯作者：
Horowitz,J
中科院分区：
文献类型：
--
作者：
Hardin,CC;Gollnick,P;Kallenbach,NR;Cohn,M;Horowitz,J
摘要：~(19)F核磁共振已用于研究具有完全活性的大肠杆菌tRNA/3‘，其中5-氟尿嘧啶取代了所有尿嘧啶及其衍生的修饰碱基的90%以上。天然tRNA的19F谱包含所有14个掺入的5-氟尿嘧啶的分辨共振。这些药物分布在6ppm的范围内，从标准的游离5-氟尿嘧啶的1.8到7.7ppm的下场。~(19)F共振是tRNA构象的灵敏监测器。去除镁或添加氯化钠会在~(19)F谱中产生重大的可逆变化。受影响最大的是天然tRNA光谱中最低的场共振(A峰)。当镁离子浓度降低或氯化钠浓度升高时，这会使磁场前移2-3ppm。TRNA的热变性导致光谱崩塌到以4.7ppm为中心的单一宽峰。对~(19)F谱图的pH依赖性研究表明，~(19)F信号位于光谱中心4-5.5ppm的五个被掺入的氟尿嘧啶，峰C、D、E、F和H可在pH 4.5-9范围内滴定。它们的p^a都接近游离的5-氟尿嘧啶(约7.5)。还提供了在中等酸性的PHS，约5.5，tRNA发生构象变化的证据。4个可滴定的5-氟尿嘧啶残基，对应于氟标记tRNA^31的19F谱中的D、E/F和H峰，由tRNA从H20转移到2H2G时的溶剂同位素位移(SIS)确定，基本上完全暴露在溶剂中。这些也是容易与亚硫酸氢盐形成加合物的5-氟尿嘧啶，亚硫酸氢盐是一种在单链区域优先与嘧啶反应的试剂。根据这些结果，19F谱中部的D、E、F和H共振被归因于tRNA非碱基配对(环)区域的5-氟尿嘧啶。来自19F光谱离子强度依赖性的证据和基于最近对氟化tRNA的其他研究的论点支持了先前的建议[Horowitz，J.，Ofengand，J.，Daniel，W.E.，&Cohn，M.(1977)J.Biol]。化学。]，低场共振对应于叔氢键合的5-氟尿嘧啶。考虑到环电流效应和4/-(羟甲基)-4，5‘，8-三甲基补骨脂素的环光加成对上场19F共振的优先扰动，已知这种环光加成最容易与双链区的嘧啶反应，从而允许将上场共振初始分配给螺旋茎中的5-氟尿嘧啶。结果表明，19F核磁共振有希望作为tRNA溶液结构的探针。UClear磁共振研究提供了关于tRNA溶液的结构、构象和动力学性质的丰富信息(Reid，1981；Reid&Hare，1982)。
Revised Manuscript Received May 16, 1986 abstract: 19F nuclear magnetic resonance has been used to study fully active Escherichia coli tRNA/3'in which 5-fluorouracil has replaced more than 90% of all uracil and uracil-derived modified bases. The 19F spectrum of the native tRNA contains resolved resonances for all 14 incorporated 5-fluorouracils. These are spread over a 6 ppm range, from 1.8 to 7.7 ppm downfield of the standard free 5-fluorouracil. The 19F resonances serve as sensitive monitors oftRNA conformation. Removal of magnesium or addition of NaCl produces major, reversible changes in the 19F spectrum. Most affected is the lowest field resonance (peak A) in the spectrum of the native tRNA. This shifts 2-3 ppm upfield as the Mg2+ concentration is lowered or the NaCl concentration is raised. Thermal denaturation of the tRNA results in a collapse of the spectrum to a single broad peak centered at 4.7 ppm. Study of the pH dependence of the 19F spectrum shows that five incorporated fluorouracils with 19F signals in the central, 4-5.5 ppm, region of the spectrum, peaks C, D, E, F, and H, are accessible to titration in the pH 4.5-9 range. All have p^ a’s close to that of free 5-fluorouridine (ca. 7.5). Evidence for a conformation change in the tRNA at mildly acidic pHs, ca. 5.5, is also presented. Four of the titratable 5-fluorouracil residues, those corresponding to peaks D, E/F, and H in the 19F spectrum of fluorine-labeled tRNA^ 31, are essentially completely exposedto solvent as determined by the solvent isotope shift (SIS) on transfer of the tRNA from H20 to 2H2G. These are also the 5-fluorouracils that readily form adducts with bisulfite, a reagent that reacts preferentially with pyrimidines in single-stranded regions. On the basis of these results, resonances D, E, F, and H in the middle of the 19F spectrum are attributed to 5-fluorouracils innon-base-paired (loop) regions of the tRNA. Evidence from the ionic strength dependence of the 19F spectrum and arguments based on other recent studies with fluorinated tRNAs support earlier suggestions [Horowitz, J., Ofengand, J., Daniel, W. E., & Cohn, M.(1977) J. Biol. Chem. 252, 4418-4420] that the resonances at lowest field correspond to tertiary hydro-gen-bonded 5-fluorouracils. Consideration of ring-current effects and the preferential perturbation of upfield 19F resonances by the cyclophotoaddition of 4/-(hydroxymethyl)-4, 5', 8-trimethylpsoralen, which is known to react most readily with pyrimidines in double-strandedregions, permits initial assignment of upfield resonances to 5-fluorouracils in helical stems. The results demonstrate the promise of 19F nuclear magnetic resonance as a probe for the structure of tRNA insolution. uclear magnetic resonance studies have provided a wealth of information on the structure, conformation, and dynamic properties of tRNA insolution (Reid, 1981; Reid & Hare, 1982).