Correlations between fluorine-19 nuclear magnetic resonance chemical shift and the secondary and tertiary structure of 5-fluorouracil-substituted tRNA.
Correlations between fluorine-19 nuclear magnetic resonance chemical shift and the secondary and tertiary structure of 5-fluorouracil-substituted tRNA.
复制标题
氟19核磁共振化学位移与5-氟尿嘧啶取代的tRNA二级和三级结构之间的相关性。
DOI：
10.1016/0022-2836(92)90529-s
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发表时间：
1992
影响因子：
5.6
通讯作者：
Horowitz,J
中科院分区：
文献类型：
--
作者：
Chu,WC;Kintanar,A;Horowitz,J
为了完成5-氟尿嘧啶取代的大肠杆菌RNAVal的19 F核磁共振（NMR）谱的归属，确定了5-氟尿嘧啶残基参与三级相互作用的共振。由于这些分配不能直接通过用于分配5-氟尿嘧啶残基在环和茎区域的tRNA的碱基置换方法，替代分配策略。通过19 F同源性Overhauser实验鉴定了FU 54和FU 55，然后通过比较它们的19 F NMR谱与5-氟尿嘧啶标记的酵母tRNA突变体的19 F NMR谱进行归属，其中FU 54被腺嘌呤取代，FU 55被胞嘧啶取代。FU 8和FU 12是从tRNAVal突变体的19 F NMR谱中指定的，其中碱基三联体G9-C23-G12取代了野生型A9-A23-FU 12。虽然保守的U8（FU 8）被A或C取代会破坏tRNAVal的三级结构，但它对缬氨酰-tRNA合成酶的催化周转数只有很小的影响，同时降低了tRNA对酶的亲和力。对5-氟尿嘧啶取代的tRNAVal光谱中所有14个共振的19 F化学位移归属的分析表明与tRNA二级和三级结构有很强的相关性。5-环区域中的氟尿嘧啶残基在光谱的中心区域产生峰，4.4至4.9 ppm（p. p.m.）低场区的游离5-氟尿嘧啶然而，来自FU 59的信号，在tRNAVal的T环中，向低场移动超过1 p. p.m.，至5.9 p. p.m.，可能是因为这种氟尿嘧啶参与了T环和D环之间的三级相互作用。当氟尿嘧啶与腺嘌呤在螺旋茎中碱基配对时，19 F的化学位移向高场移动，达到2.0至2.8 p. p.m.的范围。对于位于受体茎基部的FU 7· A66碱基对的氟，这种高场位移不太明显，这表明FU 7仅部分堆叠在连续受体茎/T-茎螺旋中的相邻G49上。一个意料之外的发现是，5-氟尿嘧啶残基与鸟嘌呤配对的19 F共振相对于氟尿嘧啶残基与A配对的19 F共振低场移动了4 - 5 p. p.m.。在所有研究的氟化tRNA的19 F NMR谱中，最远的低场峰对应于FU 55，它取代了通常在这个位置发现的保守的假尿苷。
To complete assignment of the19F nuclear magnetic resonance (NMR) spectrum of 5-fluorouracil-substitutedEscherichia colitRNAVal, resonances from 5-fluorouracil residues involved in tertiary interactions have been identified. Because these assignments could not be made directly by the base-replacement method used to assign 5-fluorouracil residues in loop and stem regions of the tRNA, alternative assignment strategies were employed. FU54 and FU55 were identified by19F homonuclear Overhauser experiments and were then assigned by comparison of their19F NMR spectra with those of 5-fluorouracil-labeled yeast tRNAPhemutants having FU54 replaced by adenine and FU55 replaced by cytosine. FU8 and FU12, were assigned from the19F NMR spectrum of the tRNAValmutant in which the base triple G9-C23-G12 substituted for the wild-type A9-A23-FU12. Although replacement of the conserved U8 (FU8) with A or C disrupts the tertiary structure of tRNAVal, it has only a small effect on the catalytic turnover number of valyl-tRNA synthetase, while reducing the affinity of the tRNA for enzyme. Analysis of the19F chemical shift assignments of all 14 resonances in the spectrum of 5-fluorouracil-substituted tRNAValindicated a strong correlation to tRNA secondary and tertiary structure. 5-Fluorouracil residues in loop regions gave rise to peaks in the central region of the spectrum, 4.4 to 4.9 parts per million (p.p.m.) downfield from free 5-fluorouracil. However, the signal from FU59, in the T-loop of tRNAVal, was shifted more than 1 p.p.m. downfield, to 5.9 p.p.m., presumably because of the involvement of this fluorouracil in the tertiary interactions between the T and D-loops. The19F chemical shift moved upfield, to the 2.0 to 2.8 p.p.m. range, when fluorouracil was base-paired with adenine in helical stems. This upfield shift was less pronounced for the fluorine of the FU7 · A66 base-pair, located at the base of the acceptor stem, an indication that FU7 is only partially stacked on the adjacent G49 in the continuous acceptor stem/T-stem helix. An unanticipated finding was that the19F resonances of 5-fluorouracil residues wobble base-paired with guanine were shifted 4 to 5 p.p.m. downfield of those from fluorouracil residues paired with A. In the19F NMR spectra of all fluorinated tRNAs studied, the farthest downfield peak corresponded to FU55, which replaced the conserved pseudouridine normally found at this position.