Felden B, Himeno H, Muto A, McCutcheon J P, Atkins J F, Gesteland R F
Howard Hughes Medical Institute, University of Utah, Salt Lake City 84112, USA.
RNA. 1997 Jan;3(1):89-103.
The conformation of the Escherichia coli 10Sa RNA (tmRNA) in solution was investigated using chemical and enzymatic probes. Single- and double-stranded domains were identified by hydrolysis of tmRNA in imidazole buffer and by lead(II)-induced cleavages. Ribonucleases T1 and S1 were used to map unpaired nucleotides and ribonuclease V1 was used to identify paired bases or stacked nucleotides. Specific atomic positions of bases were probed with dimethylsulfate, a carbodiimide, and diethylpyrocarbonate. Covariations, identified by sequence alignment with nine other tmRNA sequences, suggest the presence of several tertiary interactions, including pseudoknots. Temperature-gradient gel electrophoresis experiments showed structural transitions of tmRNA starting around 40 degrees C, and enzymatic probing performed at selected temperatures revealed the progressive melting of several predicted interactions. Based on these data, a secondary structure is proposed, containing two stems, four stem-loops, four pseudoknots, and an unstable structural domain, some connected by single-stranded A-rich sequence stretches. A tRNA-like domain, including an already reported acceptor branch, is supported by the probing data. A second structural domain encompasses the coding sequence, which extends from the top of one stem-loop to the top of another, with a 7-nt single-stranded stretch between. A third structural module containing pseudoknots connects and probably orients the tRNA-like domain and the coding sequence. Several discrepancies between the probing data and the phylogeny suggest that E. coli tmRNA undergoes a conformational change.
运用化学和酶促探针研究了溶液中大肠杆菌10Sa RNA（转运信使RNA）的构象。通过在咪唑缓冲液中水解转运信使RNA以及铅（II）诱导的切割来鉴定单链和双链结构域。核糖核酸酶T1和S1用于绘制未配对核苷酸图谱，核糖核酸酶V1用于鉴定配对碱基或堆积核苷酸。用硫酸二甲酯、碳二亚胺和焦碳酸二乙酯探测碱基的特定原子位置。通过与其他九条转运信使RNA序列进行序列比对鉴定出的共变表明存在几种三级相互作用，包括假结。温度梯度凝胶电泳实验表明转运信使RNA在约40摄氏度开始发生结构转变，在选定温度下进行的酶促探测揭示了几种预测相互作用的逐步解链。基于这些数据，提出了一种二级结构，包含两个茎、四个茎环、四个假结和一个不稳定结构域，有些通过富含腺嘌呤的单链序列片段相连。探测数据支持了一个类似tRNA的结构域，包括一个已报道的受体分支。第二个结构域包含编码序列，该序列从一个茎环的顶部延伸到另一个茎环的顶部，中间有一个7个核苷酸的单链片段。包含假结的第三个结构模块连接并可能定向类似tRNA的结构域和编码序列。探测数据与系统发育之间的几个差异表明大肠杆菌转运信使RNA发生了构象变化。
