Recognition of the universally conserved 3'-CCA end of tRNA by elongation factor EF-Tu.
Escherichia coli tRNA(Val) 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, tRNA(Val) 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-tRNA(Val):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-tRNA(Val) for EF-Tu:GTP, thus explaining the inability of these tRNA(Val) variants to function in polypeptide synthesis. Valyl-tRNA(Val) 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 tRNA(Ala) and tRNA(Phe) 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.
Liu
J C
JC
Department of Biochemistry and Biophysics, Iowa State University, Ames 50011, USA.
Liu
M
M
Horowitz
J
J
D004926:Escherichia coli; D006160:Guanosine Triphosphate / Q000378:metabolism*; D009154:Mutation; D010444:Peptide Elongation Factor Tu / Q000378:metabolism*; D011485:Protein Binding; D014176:Protein Biosynthesis; D012343:RNA, Transfer / Q000235:genetics / Q000378:metabolism*; D012344:RNA, Transfer, Ala / Q000235:genetics / Q000378:metabolism; D012346:RNA, Transfer, Amino Acyl / Q000378:metabolism*; D012360:RNA, Transfer, Phe / Q000235:genetics / Q000378:metabolism; D012366:RNA, Transfer, Val / Q000235:genetics / Q000378:metabolism
D012344:RNA, Transfer, Ala; D012346:RNA, Transfer, Amino Acyl; D012360:RNA, Transfer, Phe; D012366:RNA, Transfer, Val; D006160:Guanosine Triphosphate; D012343:RNA, Transfer; D010444:Peptide Elongation Factor Tu