Open AccessInteraction of fMet‐tRNA fMet and fMet‐AMP with the C‐terminal domain of Thermus thermophilus translation initiation factor 2
Author(s) -
Szkaradkiewicz Karol,
Zuleeg Tilman,
Limmer Stefan,
Sprinzl Mathias
Publication year - 2000
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1046/j.1432-1033.2000.01480.x
Subject(s) - thermus thermophilus , eukaryotic translation , domain (mathematical analysis) , chemistry , biology , translation (biology) , biochemistry , gene , escherichia coli , messenger rna , mathematical analysis , mathematics
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 1 H‐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.