Presence and location of modified nucleotides in Escherichia coli tmRNA:: structural mimicry with tRNA acceptor branches
Presence and location of modified nucleotides in Escherichia coli tmRNA:: structural mimicry with tRNA acceptor branches
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DOI：
10.1093/emboj/17.11.3188
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发表时间：
1998-06-01
期刊：
影响因子：
11.4
通讯作者：
Crain, PF
中科院分区：
文献类型：
--
作者：
Felden, B;Hanawa, K;Crain, PF
大肠杆菌tmRNA的功能独特的tRNA和mRNA，并具有类似于典型的tRNA的结构元件。为了测试这种模仿是否延伸到转录后修饰，结合液相色谱/电喷雾电离质谱技术，使用LC/ESIMS（LC/ESIMS）和序列数据来测定由RNase T-1水解产生的所有寡核苷酸的分子量，平均误差为0.1 Da。产生质量变化的修饰核苷酸的化学表征和序列放置。此外，化学修饰用于定位质量沉默修饰。天然大肠杆菌tmRNA含有两个修饰的核苷，5-甲基尿苷和假尿苷。这两种修饰都位于所提出的tRNA样结构域内，在模拟典型tRNA中T环保守序列的七核苷酸环中，尽管tmRNA受体分支（受体茎和T茎环）利用与典型tRNA不同的结构规则，但它们在溶液中的构象可能非常相似。通过体外转录和天然大肠杆菌tmRNA进行的未修饰tmRNA的比较结构和功能分析表明，这些转录后修饰中的一个或两个可能是有效氨酰化所需的受体分支的最佳稳定性所需的。
Escherichia coli tmRNA functions uniquely as both tRNA and mRNA and possesses structural elements similar to canonical tRNAs. To test whether this mimicry extends to post-transcriptional modification, the technique of combined liquid chromatography/ electrospray ionization mass spectrometry (LC/ESIMS) and sequence data were used to determine the molecular masses of all oligonucleotides produced by RNase T-1 hydrolysis with a mean error of 0.1 Da. Thus, this allowed for the detection, chemical characterization and sequence placement of modified nucleotides which produced a change in mass. Also, chemical modifications were used to locate mass-silent modifications. The native E.coli tmRNA contains two modified nucleosides, 5-methyluridine and pseudouridine. Both modifications are located within the proposed tRNA-like domain, in a seven-nucleotide loop mimicking the conserved sequence of T loops in canonical tRNAs, Although tmRNA acceptor branches (acceptor stem and T stem-loop) utilize different architectural rules than those of canonical tRNAs, their conformations in solution may be very similar. A comparative structural and functional analysis of unmodified tmRNA made by in vitro transcription and native E,coli tmRNA suggests that one or both of these post-transcriptional modifications may be required for optimal stability of the acceptor branch which is needed for efficient aminoacylation.