E.coliinitiator tRNA analogs with different nucleotides in the discriminator base position

Abstract
The effect of base changes at the fourth position from the 3′-terminus of Esoheriohia coli initiator tRNA Met has been studied to test the ′discriminator hypothesis′ which proposed that the nucleotide in this position might have a role in the specificity of the aminoacylation reaction. E. coli initiator tRNA lacking the 3′-terminal tetranucleotlde was prepared by partial digestion with S1 nuclease. To construct tRNA analogs with different bases in the fourth position this truncated tRNA was Joined by RNA ligase to each of four chemically synthesized 2′, 3′-ethoxy-methylidene tetranucleotides pACCA(em), pCCCA(em), pGCCA(em), and pUCCA(em). In vitro aminoacylation studies showed that all four molecules accepted methionine, albeit with different Vmax values.