MAPPING TRANSFER-RNA STRUCTURE IN SOLUTION USING DOUBLE-STRAND-SPECIFIC RIBONUCLEASE V1 FROM COBRA VENOM
MAPPING TRANSFER-RNA STRUCTURE IN SOLUTION USING DOUBLE-STRAND-SPECIFIC RIBONUCLEASE V1 FROM COBRA VENOM
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DOI：
10.1093/nar/9.19.5125
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发表时间：
1981-01-01
影响因子：
14.9
通讯作者：
KUMAR, A
中科院分区：
文献类型：
--
作者：
LOCKARD, RE;KUMAR, A
本文描述了一种用眼镜蛇毒中的双链特异性核糖核酸酶V_（11）定位延伸和起始tRNA中所有碱基配对的茎的方法。首先用双链特异性核糖核酸酶V_（11）在接近生理条件下部分消化32 p末端标记的RNA，然后在90%甲酰胺中的聚丙烯酰胺凝胶的相邻泳道中按大小对所得片段进行电泳分级。放射自显影后，通过比较V1产生的条带与脉孢菌核酸内切酶和碱基特异性核糖核酸酶产生的已知长度的片段，确定碱基配对核苷酸的位置。以酵母tRNAPe和E.在已知三维结构的大肠杆菌tRNAMet中，我们发现V1核酸酶在每个碱基配对的茎中完全切割。我们的研究还表明，核酸酶V1将消化配对的核苷酸不通过标准的沃森-克里克碱基配对氢键。在酵母中，摆动碱基对和参与三级碱基-碱基氢键的核苷酸都被切断。
A method for mapping all base-paired stems in both elongation and initiator tRNAs is described using double-strand-specific ribonuclease V1from the venom of the cobraNaja naja oxiana.32p-end-labeled RNA is first partially digested with double-strand-specific V1nuclease under near physiological conditions, and the resultant fragments are then electrophoretically fractionated by size in adjacent lanes of a polyacrylamide gel run in 90% formamide. After autoradiography, the base-paired nucleotides are definitively located by comparing V1generated bands with fragments of known length produced by bothNeurosporaendonuclease and base-specific ribonucleases. Using the substrates yeast tRNAPheand E. coli tRNAMetof known three-dimensional structure, we find V1nuclease to cleave entirely within every base-paired stem. Our studies also reveal that nuclease V1will digest paired nucleotides not hydrogen-bonded by standard Watson-Crick base-pairing. In yeast tRNAPhecleavage of both wobble base-pairs and nucleotides involved in tertiary base-base hydrogen bonding is demonstrated.