Randerath E, Gupta R C, Chia L L, Chang S H, Randerath K
Eur J Biochem. 1979 Jan 2;93(1):79-94. doi: 10.1111/j.1432-1033.1979.tb12797.x.
A second major species of leucine tRNA, tRNA Leu UAG (formerly designated tRNA Leu CUA) was purified from baker's yeast in a three-step procedure entailing BD-cellulose chromatography in the presence and absence of Mg2+ and Sephadex G-100 gel filtration. Results of aminoacylation and partial RNase T1 digestion experiments showed that this tRNA retains a native conformation under conditions that denature yeast tRNA Leu m5CAA (tRNA3 Leu). The primary structure of baker's yeast tRNA Leu UAG was elucidated by application of sensitive radioactive isotope derivative ("postlabeling") methods. Complete RNase T1 and A and partial RNase U2 fragments, prepared from non-radioactive tRNA and 5'-half and 3'-half molecules, were separated by two-dimensional polyethyleneimine-cellulose anion-exchange thin-layer chromatography and isolated by a novel micropreparative procedure affording high yields of these compounds in sufficient purity for subsequent tritium derivative analysis. Base composition and sequence of oligonucleotides were analyzed by tritium derivative methods. Molar ratios of the fragments were determined from the radioactivity of 3H-labeled nucleoside trialcohols in combination with base analysis. 2'-O-Methylated guanosine was characterized using the [gamma-32P]ATP/polynucleotide kinase reaction. The analysis of classical complete and partial RNase digests by the tritium derivative methods yielded the complete nucleotide sequence of the tRNA. A total of about 20 A260 units of the RNA was used for analysis, i.e. considerably less material than required for conventional spectrophotometric analysis. A different sequencing approach, consisting of a combination of "readout sequencing" with tritium sequencing of complete RNase T1 and A fragments, was applied to the 3'-half molecule. The 3'-half molecule was labeled with 32P at its 5' terminus, partially degraded with RNase T1, U2, and Phy1 and with alkali, and subjected to polyacrylamide gel electrophoresis. The sequence was read off the gel on the basis of cleavage patterns and size of the fragments. While the readout procedure provided only the positions of A, U, C, and G residues in the chain, additional information from tritium derivative analysis was utilized to define the positions of the modified nucleosides. The readout sequencing procedure was found to require less than 0.01 A260 unit of RNA and the analysis of the complete fragments about 6 A260 units. Interesting structural features of tRNA Leu UAG are (a) the location of unique, leucine tRNA iso-acceptor-specific sequences next to U-8, a constant nucleotide participating in synthetase recognition, (b) the occurrence of 1-methyladenosine in the T loop, a modification not present in the structurally related tRNA Leu m5CAA, and (c) the unusual presence of an unmodified uridine in the first position of the anticodon, which may be related to the unusual coding properties reported for this tRNA.
亮氨酸tRNA的第二种主要类型，即tRNA Leu UAG（以前称为tRNA Leu CUA），通过三步法从面包酵母中纯化得到，该方法包括在有和没有Mg2+的情况下进行BD-纤维素层析以及Sephadex G-100凝胶过滤。氨酰化和部分核糖核酸酶T1消化实验的结果表明，在使酵母tRNA Leu m5CAA（tRNA3 Leu）变性的条件下，这种tRNA保留了天然构象。通过应用灵敏的放射性同位素衍生物（“后标记”）方法阐明了面包酵母tRNA Leu UAG的一级结构。由非放射性tRNA以及5'-半分子和3'-半分子制备的完整核糖核酸酶T1和A片段以及部分核糖核酸酶U2片段，通过二维聚乙烯亚胺-纤维素阴离子交换薄层层析进行分离，并通过一种新颖的微量制备方法进行分离，该方法以足够的纯度提供这些化合物的高产量，用于后续的氚衍生物分析。通过氚衍生物方法分析寡核苷酸的碱基组成和序列。根据3H标记的核苷三醇的放射性结合碱基分析确定片段的摩尔比。使用[γ-32P]ATP/多核苷酸激酶反应对2'-O-甲基化鸟苷进行表征。通过氚衍生物方法对经典的完整和部分核糖核酸酶消化产物进行分析，得出了tRNA的完整核苷酸序列。总共约20个A260单位的RNA用于分析，即所需材料比传统分光光度分析少得多。一种不同的测序方法，由“读出测序”与完整核糖核酸酶T1和A片段的氚测序相结合，应用于3'-半分子。3'-半分子在其5'末端用32P标记，用核糖核酸酶T1、U2和Phy1以及碱进行部分降解，然后进行聚丙烯酰胺凝胶电泳。根据片段的切割模式和大小从凝胶上读出序列。虽然读出程序仅提供链中A、U、C和G残基的位置，但利用来自氚衍生物分析的额外信息来确定修饰核苷的位置。发现读出测序程序需要少于0.01 A260单位的RNA，而完整片段的分析需要约6 A260单位。tRNA Leu UAG有趣的结构特征是：（a）独特的亮氨酸tRNA同工受体特异性序列位于U-8旁边，U-8是参与合成酶识别的恒定核苷酸；（b）T环中存在1-甲基腺苷，这是结构相关的tRNA Leu m5CAA中不存在的修饰；（c）反密码子第一位异常存在未修饰的尿苷，这可能与该tRNA报道的异常编码特性有关。
