The Rodin-Ohno hypothesis holds that ancestral forms of Class I and II aminoacyl-tRNA synthetases (aaRS) had fully complementary nucleic acid coding sequences and that contemporary aaRS descended from opposite strands of a single gene. The schematic in A illustrates how this hypothesis leads directly to the concept of aaRS Urzymes. Antiparallel alignment of amino acid sequences for the Class-defining motifs (HIGH and KMSKS from Class I; Motifs 1 and 2 from Class II) reveals that neither anticodon-binding domain, nor a long insertion in each Class can physically be a part of such an ancestral gene. As a result, the only portions of the two Classes consistent with the hypothesis (B) are about 120-130 residues long. These fragments coincide with invariant tertiary structural cores shared by all superfamily members. Moreover, these segments together compose a minimal active site, containing binding sites for all three substrates (C). Amino acid and ATP determinants are reflected across the gene sequence, while tRNA binding determinants are related by two-fold rotation .