Complete nuclear magnetic resonance signal assignments and initial structural studies of [13C]methyl-enriched yeast transfer ribonucleic acid.
Complete nuclear magnetic resonance signal assignments and initial structural studies of [13C]methyl-enriched yeast transfer ribonucleic acid.
复制标题
富含[13C]甲基的酵母转移核糖核酸的完整核磁共振信号分配和初步结构研究。
DOI：
10.1021/bi00275a013
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发表时间：
1983
期刊：
影响因子：
2.9
通讯作者：
Schmidt,PG
中科院分区：
文献类型：
--
作者：
Agris,PF;Kovacs,SA;Smith,C;Kopper,RA;Schmidt,PG
Paul F.放大图片作者：Aubrey A，克莉丝汀史密斯，Randall A. Kopper和Paul G.施密特摘要：酵母tRNA体内碳-13甲基富集产生的探针不干扰天然tRNA结构，位于19个不同的序列位置，对RNA的结构干扰敏感。独家13 C富集甲基基团的培养基的最佳生长的培养物和最大的转录后掺入[13 C]甲基基团的蛋氨酸营养缺陷型的酿酒酵母的核酸的设计完成。从这些培养物中分离的富含碳-13的tRNA和从与[3 H]甲基甲硫氨酸一起生长的类似培养物中分离的[3 H]甲基标记的tRNA通过核苷的高效液相色谱分析被完全甲基化。发现92%的放射化学标记与甲基化核苷相关。富含13 C的tRNA的碳-13 NMR光谱在11和60 ppm之间表现出突出的高场甲基信号。相对于天然丰度（1.1%）核糖碳的信号面积的积分指示50-60原子%[13 C]甲基富集。对胸苷、5-甲基胞苷、N-甲基鸟苷、3-甲基胞苷、1-甲基鸟苷、1-甲基腺苷、7-甲基鸟苷、N-甲基鸟苷、5-（甲氧羰基甲基）尿苷以及胞苷、鸟苷和尿苷的2 ′-O-甲基衍生物的甲基碳进行了信号分配。已知这些核苷位于三叶草的环和茎区的19个序列位置上。这是由于在细胞中的量小和难以纯化。因此，大分子构象的非破坏性测定是重要的，以便保留RNA或DNA用于多种研究。核磁
Paul F. Agris,* Shirley AH Kovacs, Christine Smith, Randall A. Kopper, and Paul G. Schmidt abstract: Carbon-13 methyl enrichment of yeast tRNA in vivo has produced probes that do notperturb native tRNA structure, are located at 19 distinct sequence positions, and are sensitive to structural perturbations of the RNA. Exclusive 13C enrichment of methyl groups was accomplished by design of a medium for optimal growth of cultures and maximal posttranscriptional incorporation of [13C] methyl groups into nucleic acids of a methionine auxotroph of Saccharomyces cerevisiae. Carbon-13-enriched tRNA isolated from these cultures and [3H] methyl-labeled tRNA isolated from analogous cultures grown with [3H] methylmethionine were fully methylated as determined by high-performance liquid chromatographic analysis of nucleosides. Ninety-two percent of the radiochemical label was found associated with methylated nucleosides. Carbon-13 NMR spectra of 13C enriched tRNA exhibited prominent high-field, methyl signals between 11 and 60 ppm. Integration of signal area relative to that of the natural abundance (1.1%) ribose carbons indicated a 50-60 atom%[13C] methyl enrichment. Signal assignmentshave been made for the methyl carbons of ribothymidine, 5-methylcytidine, iV^ methylguanosine, 3-methylcytidine, 1-methylguanosine, 1-methyladenosine, 7-methylguanosine, iV^/VMimethylguanosine, 5-(methoxycarbonylmethyl) uridine, and the 2'-O-methyl derivatives of cytidine, guanosine, and uridine. These nucleosides are known to be located at 19 sequence positions in loops and stem regions of the cloverleafThe amount of specific nucleic acid available for the study of native secondary and tertiary structure in solution is re-stricted. This is due to the small amounts in cells and difficulty in purification. Thus, a nondestructive determination of macromolecular conformation is important in order to retain the RNA or DNA for multiple studies. Nuclear magnetic