Permutation of a pair of tertiary nucleotides in a transfer RNA.

Abstract

The tertiary nucleotides at positions 15 and 48 in a tRNA establish non-Watson-Crick hydrogen interactions that connect the dihydrouridine (D) loop with the variable loop and stabilize the "L"-shaped tRNA structure. Although the majority of tRNAs have G15.C48 or A15.U48, all of the 16 possible nucleotide pairs at positions 15 and 48 can be found in the existing cytoplasmic and mitochondrial tRNA sequences. Because tRNAs contain a variety of slightly different sets of tertiary nucleotides, this complexity raises the question of whether a given tRNA sequence framework can accommodate all of the 16 compositions at positions 15 and 48. In this work, G15 and C48 in an Escherichia coli alanine amber suppressor tRNA were permuted, and variants were tested for biological activity in vivo. All but an A15.A48 variant were functional, indicating substantial flexibility at positions 15 and 48 to accommodate nucleotide variations. Analysis of the A15.A48 variant with chemical probes showed that this mutant harbors a defect that specifically changes the conformation of the anticodon sequence. Interestingly, human tRNA(Ala) has A15.A48. Additional nucleotide substitutions in E. coli A15.A48 tRNA(Ala) that recreate the D loop sequence of human tRNA(Ala) restored the biological activity to this tRNA by reestablishing the wild-type conformation of the anticodon sequence. The results suggest a distal relationship between the D and the anticodon loops in a tRNA and delineate covariation of specific nucleotides in the evolution of tRNA(Ala) from E. coli to human.