The protein synthesis initiation factor 2 G-domain. Study of a functionally active C-terminal 65-kilodalton fragment of IF2 from Escherichia coli.
Protein synthesis initiation factor 2 (IF2) is present in Escherichia coli cells as two forms which are expressed from the same gene: IF2 alpha [97.3 kilodaltons (kDa)] and IF2 beta (79.7 kDa). During isolation, a smaller form, IF2 gamma, is generated, presumably by partial proteolysis. It has been purified to homogeneity and has an apparent mass of 70 kDa, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Immunoelectrophoresis of IF2 alpha and IF2 gamma shows that IF2 gamma is immunologically partially identical with IF2 alpha. The sequence of the 15 N-terminal amino acid residues of IF2 gamma was determined and compared with that of IF2 alpha. The N-terminal amino acid of IF2 gamma corresponds to Arg-290 of IF2 alpha, suggesting that IF2 gamma is generated by proteolytic cleavage of the Lys-289-Arg-290 bond of IF2. Assuming a C terminus identical with IF2 alpha, we calculate that IF2 gamma comprises 601 amino acid residues and has a mass of 64.8 kDa. The truncated protein was tested for activities characteristic of IF2 in three in vitro assays: fMet-tRNA(fMet) binding to 70S ribosomes, N-terminal dipeptide synthesis in a DNA-dependent transcription/translation system, and ribosome-dependent GTP hydroly97-7. The specific activities of IF2 gamma were comparable with, or only slightly less than, those for IF2 alpha, indicating that IF2 gamma contains the active centers for interaction with fMet-tRNA(fMet), ribosomes, and GTP. A central region in the primary structure of IF2 shows extensive sequence homology with a number of GDP-binding proteins and especially with the G-domain of elongation factor Tu (EF-Tu).(ABSTRACT TRUNCATED AT 250 WORDS)
D000595:Amino Acid Sequence; D004151:Dipeptides / Q000096:biosynthesis; D000939:Epitopes / Q000032:analysis; D004926:Escherichia coli / Q000235:genetics*; D015852:Eukaryotic Initiation Factor-2; D020558:GTP Phosphohydrolases / Q000378:metabolism; D007700:Kinetics; D008970:Molecular Weight; D010446:Peptide Fragments / Q000302:isolation & purification; D010448:Peptide Initiation Factors / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism*; D011487:Protein Conformation; D011506:Proteins / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism*; D012689:Sequence Homology, Nucleic Acid
D004151:Dipeptides; D000939:Epitopes; D015852:Eukaryotic Initiation Factor-2; D010446:Peptide Fragments; D010448:Peptide Initiation Factors; D011506:Proteins; D020558:GTP Phosphohydrolases