Studies on T. utilis tRNATyr variants with enzymatically altered D-loop sequences. II. Relationship between the...

The nucleotide sequence of T. utilis tRNATyr has been modified to have a deletion or substitution of the "conserved" nucleotide sequence Gm18-G19 in the D-loop by enzymatic procedures in vitro. Conformations of the variant tRNAs were analyzed by measuring melting profiles and electrophoretic mobilities in "native" polyacrylamide gels, and by examining the RNase T1 digestion patterns in sequencing gels. The results obtained shed light on the importance of the interaction between the sequence Gm18-G19 and nucleotides in the T psi C-loop (probably psi 57-C58) for the maintenance of the total conformation of tRNATyr in solution. The association of D-loop and T psi C-loop regions in the variant tRNATyrs is slightly relaxed even at room temperature and melting occurred at temperatures higher than 40 degrees C. The relationship between the tertiary structure of the variant tRNA and its aminoacylation capacity was assayed at various temperatures. The results indicate that highly ordered tertiary structure is needed for tRNATyr to be fully aminoacylated.