Interaction of fMet-tRNAfMet and fMet-AMP with the C-terminal domain of Thermus thermophilus translation initiation factor 2

Two polypeptides resistant against proteolytic digestion were identified in Thermus thermophilus translation initiation factor 2 (IF2): the central part of the protein (domains II/III), and the C-terminal domain (domain IV). The interaction of intact IF2 and the isolated proteolytic fragments with fMet-tRNA(fMet) was subsequently characterized. The isolated C-terminal domain was as effective in binding of the 3' end of fMet-tRNA(f Met) as intact IF2. N-Formylation of Met-tRNA(fMet) was required for its efficient binding to the C-terminal domain. This suggests that the interaction between the C-terminal domain and the 3' end of fMet-tRNA(fMet) is responsible for the recognition of fMet-tRNA(fMet) by IF2 during translation initiation. Moreover, it was demonstrated that fMet-AMP is a minimal ligand of IF2. fMet-AMP inhibits fMet-tRNA(fMet) binding to IF2 as well as the activity of IF2 in the stimulation of ApUpG-dependent ribosomal binding of fMet-tRNA(f Met). Specific interaction of fMet-AMP with IF2 was demonstrated by H-1-NMR spectroscopy. These findings indicate that fMet-AMP and the 3' terminal fMet-adenosine of fMet-tRNA(fMet) use the same binding site on the C-terminal domain of IF2 and imply that the interaction between the C-terminal domain and the 3' end of fMet-tRNA(fMet) is primarily responsible for the fMet-tRNA(fMet) binding and recognition by IF2.