Mapping the fMet-tRNA(f)(Met) binding site of initiation factor IF2.
The interaction between fMet-tRNA(f)(Met) and Bacillus stearothermophilus translation initiation factor IF2 has been characterized. We demonstrate that essentially all thermodynamic determinants governing the stability and the specificity of this interaction are localized within the acceptor hexanucleotide fMet-3'ACCAAC of the initiator tRNA and a fairly small area at the surface of the beta-barrel structure of the 90-amino acid C-terminal domain of IF2 (IF2 C-2). A weak but specific interaction between IF2 C-2 and formyl-methionyl was also demonstrated. The surface of IF2 C-2 interacting with fMet-tRNA(f)(Met) has been mapped using two independent approaches, site- directed mutagenesis and NMR spectroscopy, which yielded consistent results. The binding site comprises C668 and G715 located in a groove accommodating the methionyl side-chain, R700, in the vicinity of the formyl group, Y701 and K702 close to the acyl bond between fMet and tRNA(f)(Met), and the surface lined with residues K702-S660, along which the acceptor arm of the initiator tRNA spans in the direction 3' to 5'.
Guenneugues
M
M
Bijvoet Center for Biomolecular Research, Utrecht University, Padualaan 8, 3584 CH Utrecht, The Netherlands.
Caserta
E
E
Brandi
L
L
Spurio
R
R
Meunier
S
S
Pon
C L
CL
Boelens
R
R
Gualerzi
C O
CO
D001665:Binding Sites; D001411:Geobacillus stearothermophilus / Q000737:chemistry* / Q000378:metabolism; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009239:N-Formylmethionine / Q000737:chemistry / Q000378:metabolism; D010448:Peptide Initiation Factors / Q000737:chemistry* / Q000378:metabolism; D039881:Prokaryotic Initiation Factor-2; D014176:Protein Biosynthesis; D011487:Protein Conformation; D012358:RNA, Transfer, Met / Q000737:chemistry* / Q000378:metabolism; D013816:Thermodynamics
D010448:Peptide Initiation Factors; D039881:Prokaryotic Initiation Factor-2; D012358:RNA, Transfer, Met; D009239:N-Formylmethionine