Partial assignment of resonances in the 19F nuclear magnetic resonance spectra of 5-fluorouracil-substituted transfer RNAs.
Partial assignment of resonances in the 19F nuclear magnetic resonance spectra of 5-fluorouracil-substituted transfer RNAs.
复制标题
5-氟尿嘧啶取代的转移RNA的19F核磁共振谱中共振的部分分配。
DOI：
10.1021/bi00401a070
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发表时间：
1988
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;Horowitz,J
Department of Biochemistry and Biophysics，爱荷华州州立大学，艾姆斯，爱荷华州50011接收于1986年12月26日;修订的Mandarin pt接收于1987年9月9日摘要：比较了三种纯化的5-氟尿嘧啶-（FUra-）取代的大肠杆菌tRNA，tRNA 7al，tRNA 7 et和tRNAf 1 ® 1的19 F核磁共振（NMR）谱的特征。每种tRNA物质可被分解成两种同受体形式，A和B，其19 F NMR谱的不同之处在于，在同受体B的高场谱中，一个峰从4.5至4.8 ppm（FUra=0）的范围移位至约-100 ppm。15 ppm。由于每种tRNA的两种同种受体的序列仅在D环中的一个位置不同，该位置通常被二氢尿苷残基占据，因此我们将氟标记的tRNA α 1光谱中的4.5 ppm峰归属于FUra 17，将氟尿嘧啶取代的tRNA α 1光谱中的4.6 ppm共振归属于FUra 20。在19 F标记的tRNA α 1的19 F NMR谱中，在低场峰A和B之间观察到19 F（19 F）核奥弗豪泽效应的倒数。假设氟标记的tRNA α 1具有与酵母tRNAPhe类似的结构，则只有FUra 54和FUra 55足够接近（4-5 A）以产生可感知的19 F同源奥弗豪泽效应。因此，峰A和B归属于FUra 54和-55。当温度从30 ℃升高到45 ℃时，19 F标记的tRNA α 1光谱中的峰B（6.6 ppm）的强度逐渐向高场移动到6.4 ppm（Tm= 36 ℃），表明相应的5-氟尿嘧啶残基在两种磁性不同的环境之间发生了温度依赖性的缓慢交换。由于这种效应类似于几种天然tRNA光谱中T54甲基H和13 C信号的分裂[Kastrup，RV，&施密特，P. G.（1978）Nucleic Acids Res.5，257-269; Kopper，R.一、施密特，P.G.，& Agris，PF（1983）Biochemistry 22，1396-1401]，我们将峰B归属于FUra 54。19 F标记tRNA光谱中的峰A|然后可以将a1分配给FUra 55。三种tRNA中的每一种的19 F光谱中的最低场共振（峰A）随着离子强度或镁离子浓度的变化而表现出独特的大化学位移变化。这种相似性表明，峰A对应于tRNA中的保守碱基，并且与所有三种氟化tRNA的19 F NMR谱中峰A被指定为取代不变式的5-氟尿嘧啶残基^[55]相一致。
Department of Biochemistry and Biophysics, Iowa State University, Ames, Iowa 50011 Received December 26, 1986; Revised Manuscript Received September 9, 1987 abstract: Features of the 19F nuclear magnetic resonance (NMR) spectra of three purified 5-fluorouracil-(FUra-) substituted Escherichia coli tRNAs, tRNA7al, tRNA^ et, and tRNAf1® 1, are compared. Each of the tRNA species can be resolved into two isoaccepting forms, A and B, whose 19F NMR spectra differ in the shift of one peak from the 4.5 to 4.8 parts per million (ppm) range (FUra=0) in the spectrum of isoacceptor B upfield to ca.-15 ppm in that of isoacceptor A. Because the sequences of the two isoacceptors of each tRNA differ only at one position in the D loop, that normally occupied by a dihydrouridine residue, we assign the 4.5 ppm peak in the spectrum of fluorine-labeled tRNA^ al to FUra 17 and the resonance at 4.6 ppm in the spectrum of fluorouracil-substituted tRNA^ et to FUra20. A reciprocal 19F {19F) nuclear Overhauser effect is observed between the downfield peaks A and B in the 19F NMR spectrum of 19F-labeled tRNA^ al. Assuming that fluorine-labeled tRNA/al has a structure similar to that of yeast tRNAPhe, only FUra54 and-55 are close enough (4-5 A) to give an appreciable 19F homonuclear Overhauser effect. Peaks A and B have therefore been assigned to FUra54 and-55. As the temperature is raised from 30 to 45 C, the intensity of peak B (6.6 ppm) in the spectrum of 19F-labeled tRNA^ al graduallyshifts upfield to 6.4 ppm (Tm= 36 C), indicating a temperature-dependent slow exchange of the corresponding 5-fluorouracil residue between two magneticallydistinct environments. Because this effect resembles the splitting of T54 methyl'H and 13C signals in the spectra of several native tRNAs [Kastrup, RV, & Schmidt, P. G.(1978) Nucleic Acids Res. 5, 257-269; Kopper, R. A., Schmidt, P. G., & Agris, PF (1983) Biochemistry 22, 1396-1401], we assign peak B to FUra54. Peak A in the spectrum of 19F-labeled tRNA| al can then be assigned to FUra55. The lowest field resonance(peak A) in the 19F spectra of each of the three tRNAs exhibits a uniquely large chemical shift change with changing ionic strength or magnesium ion concentration. This similarity suggests that peak A corresponds to a conserved base in the tRNAs and is consistent with assignment of peak A in the 19F NMR spectra of all three fluorinated tRNAs to the 5-fluorouracil residue that replaces the invariant^ 55.