Substitution of the 3′ terminal adenosine residue of transfer RNA in vivo

We have altered by site-directed mutagenesis the 3′ terminal adenosine residue of a tRNA^Tyrsu₃⁺ gene encoded on a single-copy plasmid and examined the consequences of these substitutions on suppressor activity in vivo. Our data show that mutant su₃ genes containing 3′-CCC, -CCG, or -CCU termini instead of -CCA can be efficiently transcribed and processed in Escherichia coli to generate functional suppressor tRNAs. However, in contrast to normal tRNA genes, both tRNA nucleotidyltransferase and exoribonuclease activities are required to obtain suppression by the mutant tRNAs, indicating that removal of the incorrect 3′ terminal residue and resynthesis of the normal -CCA terminus are occurring in this situation. In addition, a low level of suppressor activity and tRNA repair was found in cells devoid of tRNA nucleotidyltransferase, suggesting that an additional activity able to partially repair the 3′ end of tRNA is present in E. coli. The use of mutant strains lacking one or several exoribonucleases revealed that the various RNases have very different specificities for removal of incorrect 3′ residues and that these differ greatly from their action on CCA-ending tRNA. These data show that the 3′ terminal adenosine residue is necessary for tRNA function in vivo and that cells can compensate for its alteration by changes in the normal pathway of tRNA metabolism.