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-tRNAfMet was subsequently characterized. The isolated C-terminal domain was as effective in binding of the 3' end of fMet-tRNAf Met as intact IF2. N-Formylation of Met-tRNAfMet 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-tRNAfMet is responsible for the recognition of fMet-tRNAfMet by IF2 during translation initiation. Moreover, it was demonstrated that fMet-AMP is a minimal ligand of IF2. fMet-AMP inhibits fMet-tRNAfMet binding to IF2 as well as the activity of IF2 in the stimulation of ApUpG-dependent ribosomal binding of fMet-tRNAf Met. Specific interaction of fMet-AMP with IF2 was demonstrated by 1H-NMR spectroscopy. These findings indicate that fMet-AMP and the 3' terminal fMet-adenosine of fMet-tRNAfMet 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-tRNAfMet is primarily responsible for the fMet-tRNAfMet binding and recognition by IF2.
Szkaradkiewicz
K
K
Laboratorium für Biochemie, Universität Bayreuth, Germany.
Zuleeg
T
T
Limmer
S
S
Sprinzl
M
M
D000249:Adenosine Monophosphate / Q000378:metabolism*; D000595:Amino Acid Sequence; D001665:Binding Sites; D015852:Eukaryotic Initiation Factor-2 / Q000378:metabolism*; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D009239:N-Formylmethionine / Q000378:metabolism*; D014176:Protein Biosynthesis; D012358:RNA, Transfer, Met / Q000378:metabolism*; D013696:Temperature; D016963:Thermus thermophilus / Q000378:metabolism*
D015852:Eukaryotic Initiation Factor-2; D012358:RNA, Transfer, Met; C024988:tRNA, formylmethionine-; D000249:Adenosine Monophosphate; D009239:N-Formylmethionine