The Rodin-Ohno hypothesis postulates that two classes of aminoacyl-tRNA synthetases were encoded complementary to double-stranded DNA. Particularly, Geobacillus stearothermophilus tryptophanyl-tRNA synthetase (TrpRS, belonging to class I) and Escherichia coli histidyl-tRNA synthetase (HisRS, belonging to class II) show high complementarity of the middle base of the codons in the mRNA sequence encoding each ATP binding site. Here, for the reported 46-residue peptides designed from the three-dimensional structures of TrpRS and HisRS, amino acid activation analysis was performed using the malachite green assay, which detects the pyrophosphate departing from ATP in the forward reaction of the first step of tRNA aminoacylation. A maltose-binding protein fusion with the 46 residues of TrpRS (TrpRS46mer) exhibited high activation capacity for several amino acids in the presence of ATP and amino acids, but the activity of an alanine substitution mutant of the first histidine in the HIGH motif (TrpRS46merH15A) was largely reduced. In contrast, pyrophosphate release by HisRS46mer in the histidine activation step was lower than that in the case of TrpRS46mer. Both HisRS46mer and the alanine mutant at the 113rd arginine (HisRS46merR113A) showed slightly higher levels of pyrophosphate release than the maltose-binding protein alone. These results do not rule out the Rodin-Ohno hypothesis, but may suggest the necessity of establishing unique evolutionary models from different perspectives.

Rodin-Ohno 假说假定两类氨酰-tRNA 合成酶被编码为与双链 DNA 互补。特别是嗜热脂肪地芽孢杆菌色氨酸-tRNA合成酶（TrpRS，属于I类）和大肠杆菌组氨酰-tRNA 合成酶（HisRS，属于 II 类）在编码每个 ATP 结合位点的 mRNA 序列中显示出密码子中间碱基的高度互补性。在这里，对于报告的从TrpRS和HisRS的三维结构设计的46个残基肽，使用孔雀石绿测定法进行氨基酸活化分析，该测定法检测在tRNA的第一步正向反应中离开ATP的焦磷酸盐氨酰化。与 TrpRS 的 46 个残基融合的麦芽糖结合蛋白 (TrpRS46mer) 在 ATP 和氨基酸存在下表现出对几个氨基酸的高活化能力，但 HIGH 基序中第一个组氨酸的丙氨酸取代突变体 (TrpRS46merH15A) 的活性) 大大减少。相比之下，HisRS46mer 在组氨酸活化步骤中的焦磷酸释放低于 TrpRS46mer 的情况。HisRS46mer 和第 113 个精氨酸的丙氨酸突变体 (HisRS46merR113A) 的焦磷酸释放水平略高于单独的麦芽糖结合蛋白。这些结果不排除罗丹-大野假说，但可能表明有必要从不同的角度建立独特的进化模型。