THE NUCLEOTIDE IN POSITION-32 OF THE TRANSFER-RNA ANTICODON LOOP DETERMINES ABILITY OF ANTICODON UCC TO DISCRIMINATE AMONG GLYCINE CODONS

We have investigated the influence of structures in the tRNA anticodon loop and stem on the ability of the anticodon to discriminate among c odons. We had previously shown that anticodon UCC, when placed in the structural context of tRNA1Gly from Escherichia coli, discriminated ef ficiently between the glycine codons, as required by the wobble rules. Thus, this anticodon read GGA and GGG but did not read GGU and GGC, w hereas in mycoplasma tRNA(Gly), the same anticodon did not discriminat e among the glycine codons. We have now determined the reading propert ies of three constructions based on tRNA1Gly containing the anticodon UCC in different structural contexts. In one of these constructs, tRNA 1Gly-ASL, the anticodon loop and stem are the same as in mycoplasma tR NA(Gly). The second construct, tRNA1Gly-AS, has an anticodon stem iden tical with the mycoplasma tRNA(Gly), whereas in the last construct, tR NA1Gly-C32, the only difference from tRNA1Gly(UCC) is that the undine in position 32 of the anticodon loop has been replaced by cytidine. Th ese constructs were tested for ability to read glycine codons in an in vitro protein-synthesizing system that allowed us to monitor separate ly the reading of each codon. We found that the anticodon UCC, when pr esent in tRNA1Gly-AS, discriminated among the glycine codons, whereas in the constructs tRNA1Gly-ASL and tRNA1Gly-C32, the same anticodon ha d lost its ability to discriminate-i.e., it behaved as in mycoplasma t RNA(Gly). These results strongly suggest that nt 32 of the anticodon l oop of tRNA1Gly(UCC) decisively influences the reading properties of t he anticodon UCC.