Hegg L A, Thurlow D L
Department of Chemistry, Clark University, Worcester, MA 01610.
Nucleic Acids Res. 1990 May 25;18(10):2993-3000. doi: 10.1093/nar/18.10.2993.
Fifteen individual species of tRNA were treated with the chemical modifiers diethylpyrocarbonate, 50% aqueous hydrazine or hydrazine/3 M NaCl. Following purification of the chemically modified material on polyacrylamide gels containing 8 M urea, variant minor bands, in addition to the expected main band, were observed for 12 of the 15 tRNAs. Characterization of the content of chemically altered bases in material recovered from such bands indicated that tRNAs containing modified nucleotides in base-paired stems were excluded from the main band and present, often in enhanced amounts, in the minor variant bands. The persistence of residual secondary structure on 8 M urea gels run at 45 degrees C and the ability of chemically modified bases to alter electrophoretic mobilities warrant caution in designing and interpreting experiments in which chemically modified RNA is isolated on gels prior to further analysis. tRNA(Val) (VAC) was unique in that modified bases in non base-paired regions, according to the cloverleaf model of secondary structure, caused exclusion from the main band. Consequently, we propose a secondary structure for partially denatured tRNA(Val) (VAC), in which these bases are located in double stranded regions of the molecule.
15种不同的tRNA用化学修饰剂焦碳酸二乙酯、50%水合肼或水合肼/3M氯化钠进行处理。在含8M尿素的聚丙烯酰胺凝胶上对化学修饰材料进行纯化后，15种tRNA中的12种除了预期的主带外还观察到了变异的次要条带。对从此类条带回收的材料中化学改变碱基的含量进行表征表明，在碱基配对茎中含有修饰核苷酸的tRNA被排除在主带之外，并经常以增加的量存在于次要变异条带中。在45℃运行的8M尿素凝胶上残留二级结构的持久性以及化学修饰碱基改变电泳迁移率的能力，在设计和解释在凝胶上分离化学修饰RNA以进行进一步分析之前的实验时需要谨慎。tRNA(Val)(VAC)是独特的，根据二级结构的三叶草模型，非碱基配对区域中的修饰碱基导致其被排除在主带之外。因此，我们提出了部分变性的tRNA(Val)(VAC)的二级结构，其中这些碱基位于分子的双链区域。
