Terminal oxidation-reduction of yeast phenylalanine tRNA prevents donor and acceptor function at the peptidyl transferase center

Periodate oxidation of the ribose of the 3′-terminal adenosine of yeast tRNAPhe followed by borohydride reduction has the net effect of splitting the C2′C3′ bond leaving two primary alcohol groups at these carbon atoms. This modified tRNA (tRNAox-red) could be acylated with phenylalanine but could not function as either a donor or acceptor at the peptidyl transferase center of the ribosome. Assays were performed with the phenylalanyl-pentanucleotides, CACCAox-red(acetylPhe) and CACCAox-red(Phe), which were isolated from the 3′-end of appropriately esterified tRNAox-red. Adoox-red(Phe) isolated from Phe-tRNAox-red was also inactive as an acceptor, but synthetic Adoox-red(Phe), a mixture of the 2′ and 3′ phenylalanyl esters, was active with an apparent Km of 1.16 mM compared to 0.2 mM for control Ado(Phe). These results are interpreted to mean that (1) biosynthetic aminoacylation of tRNAox-red occurs specifically at the 2′-hydroxyl, (2) there is no 2′:3′ tautomerization in the ring-opened structure, and (3) peptidyl transferase recognizes specifically the 3′-aminoacyl esters of tRNA.