DISTINCTIVE ACCEPTOR-END STRUCTURE AND OTHER DETERMINANTS OF ESCHERICHIA-COLI TRNA(PRO) IDENTITY

The previously uncharacterized determinants of the specificity of tRNA (Pro) for aminoacylation (tRNA(Pro) identity) were defined by a comput er comparison of all Escherichia coil tRNA sequences and tested by a f unctional analysis of amber suppressor tRNAs in vivo. We determined th e amino acid specificity of tRNA by sequencing a suppressed protein an d the aminoacylation efficiency of tRNA by examining the steady-state level of aminoacyl-tRNA. On substituting nucleotides derived from the acceptor end and variable pocket of tRNA(Pro) for the corresponding nu cleotides in a tRNA(Phe) gene, the identity of the resulting tRNA chan ged substantially but incompletely to that of tRNA(Pro). The redesigne d tRNA(Phe) was weakly active and aminoacyl-tRNA was not detected. Eth yl methanesulfonate mutagenesis of the redesigned tRNA(Phe) gene produ ced a mutant with a wobble pair in place of a base pair in the end of the acceptor-stem helix of the transcribed tRNA. This mutant exhibited both a tRNA(Pro) identity and substantial aminoacyl-tRNA. The results speak for the importance of a distinctive conformation in the accepto r-stem helix of tRNA(Pro) for aminoacylation by the prolyl-tRNA synthe tase. The anticodon also contributes to tRNA(Pro) identity but is not necessary in vivo.