Escherichia coli tRNAVal with
pyrimidine substitutions for the universally conserved
3′-terminal adenine can be readily aminoacylated.
It cannot, however, transfer valine into polypeptides.
Conversely, despite being a poor substrate for valyl-tRNA
synthetase, tRNAVal with a 3′-terminal
guanine is active in in vitro polypeptide synthesis. To
better understand the function of the 3′-CCA sequence
of tRNA in protein synthesis, the effects of systematically
varying all three bases on formation of the Val-tRNAVal:EF-Tu:GTP
ternary complex were investigated. Substitutions at C74
and C75 have no significant effect, but replacing A76 with
pyrimidines decreases the affinity of valyl-tRNAVal
for EF-Tu:GTP, thus explaining the inability of these tRNAVal
variants to function in polypeptide synthesis. Valyl-tRNAVal
terminating in 3′-guanine is readily recognized by
EF-TU:GTP. Dissociation constants of the EF-Tu:GTP ternary
complexes with valine tRNAs having nucleotide substitutions
at the 3′ end increase in the order adenine <
guanine < uracil; EF-Tu has very little affinity for
tRNA terminating in 3′ cytosine. Similar observations
were made in studies of the interaction of 3′ end
mutants of E. coli tRNAAla and tRNAPhe
with EF-Tu:GTP. These results indicate that EF-Tu:GTP preferentially
recognizes purines and discriminates against pyrimidines,
especially cytosine, at the 3′ end of aminoacyl-tRNAs.