SECONDARY STRUCTURE OF THE CIRCULAR FORM OF THE TETRAHYMENA RIBOSOMAL-RNA INTERVENING SEQUENCE - A TECHNIQUE FOR RNA STRUCTURE-ANALYSIS USING CHEMICAL PROBES AND REVERSE-TRANSCRIPTASE
SECONDARY STRUCTURE OF THE CIRCULAR FORM OF THE TETRAHYMENA RIBOSOMAL-RNA INTERVENING SEQUENCE - A TECHNIQUE FOR RNA STRUCTURE-ANALYSIS USING CHEMICAL PROBES AND REVERSE-TRANSCRIPTASE
复制标题
DOI：
10.1073/pnas.82.3.648
复制
发表时间：
1985-01-01
影响因子：
11.1
通讯作者：
CECH, TR
中科院分区：
文献类型：
--
作者：
INOUE, T;CECH, TR
四膜虫rRNA前体的干涉序列(IVS)的结构介导切割-连接反应，导致Pre-rRNA剪接和IVS环化。建立了一种RNA结构分析方法，并将其应用于IVS RNA的环状结构。用硫酸二甲酯或焦碳酸二乙酯处理天然RNA来修饰不参与二次或三次相互作用的碱基。然后将该RNA用作反转录的模板。合成的寡聚脱氧核苷酸引物的延伸被发现在1-甲基腺苷、3-甲基胞苷和7-乙氧基甲基腺苷残基之前停止(或暂停)1个核苷酸。1-甲基腺苷的检测对于定位单链区域特别有用。RNA经化学裂解后，也可检测到7-甲基鸟苷。这些修饰位点与先前关于IVS RNA线性形式的二级结构的模型一致，该模型基于酶切割数据、自由能计算和系统发育比较。IVS RNA的自环化不涉及二级结构的重大重排，尽管有证据表明分子的一个区域发生了构象变化。这里描述的方法对于获得远离RNA分子末端的结构信息应该是通用的。
The structure of the intervening sequence (IVS) of the Tetrahymena rRNA precursor mediates cleavage-ligation reactions that result in pre-rRNA splicing and IVS cyclization. A method was developed for RNA structure analysis and applied it to the circular form of the IVS RNA. The native RNA was treated with dimethyl sulfate or diethyl pyrocarbonate to modify bases not involved in secondary or tertiary interactions. The RNA was then used as a template for reverse transcription. Elongation of synthetic oligodeoxynucleotide primers was found to stop (or pause) 1 nucleotide prior to 1-methyladenosine, 3-methylcytidine and 7-ethoxycarbonyladenosine residues. The detection of 1-methyladenosine is particularly useful for locating single-stranded regions. After chemical cleavage of the RNA, 7-methylguanosine also could be detected. The sites of modification were consistent with a previous model of the secondary structure of the linear form of the IVS RNA, a model based on enzymatic cleavage data, free energy calculations and phylogenetic comparison. IVS RNA autocyclization does not involve major rearrangements of the secondary structure, although there is evidence for a conformational change in one region of the molecule. The methods described here should be of general use for obtaining information about structure far from the ends of RNA molecules.