通过在特定位置富集碳-13来制备tRNA结构的特定位点探针。

Production of specific site probes of tRNA structure by enrichment with carbon 13 at particular locations.

作者信息

Tompson J G, Agris P F

出版信息

Nucleic Acids Res. 1979 Oct 10;7(3):765-79. doi: 10.1093/nar/7.3.765.

DOI:10.1093/nar/7.3.765

原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC328054/

Abstract

Escherichia coli C6 rel met cys was cultured in a stringently defined minimal medium containing 13C-enriched metabolites in order to (1) achieve maximal 13C isotopic enrichment of tRNA; and (2) produce site specific but natural, non-perturbing NMR probes of tRNA structure and function. Growth conditions were manipulated to achieve optimal culture growth concomitant with maximal in vivo incorporation of various 13C-enriched nucleic acid precursors, including L-[methyl-13C] methionine, [2-(13)C] adenine, and [2-(13)C] uracil. Effective blockage of purine biosynthesis de novo was accomplished with the addition of the antimetabolite 6-mercaptopurine to the growth medium. Transfer RNAs specifically 13C-enriched in all methyl groups (57 atom %), C2 of adenine (60 atom %), and C2 of uracil (82 atom %) and C2 of cytosine (73 atom %) have been produced.

摘要

大肠杆菌C6 rel met cys在含有13C富集代谢物的严格定义的基本培养基中培养，目的是：（1）实现tRNA的最大13C同位素富集；（2）产生tRNA结构和功能的位点特异性但天然的、无干扰的NMR探针。通过控制生长条件，实现最佳的培养生长，同时使各种13C富集的核酸前体在体内最大程度地掺入，包括L-[甲基-13C]甲硫氨酸、[2-(13)C]腺嘌呤和[2-(13)C]尿嘧啶。通过向生长培养基中添加抗代谢物6-巯基嘌呤，有效地阻断了嘌呤的从头生物合成。已经产生了在所有甲基（57原子%）、腺嘌呤的C2（60原子%）、尿嘧啶的C2（82原子%）和胞嘧啶的C2（73原子%）中特异性13C富集的转移RNA。

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