Both forms of translational initiation factor IF2 (alpha and beta) are required for maximal growth of Escherichia coli. Evidence for two translational initiation codons for IF2 beta.
The gene infB codes for two forms of translational initiation factor IF2; IF2 alpha (97,300 Da) and IF2 beta (79,700 Da). IF2 beta arises from an independent translational event on a GUG codon located 471 bases downstream from IF2 alpha start codon. By site-directed mutagenesis we constructed six different mutations of this GUG codon. In all cases, IF2 beta synthesis was variably affected by the mutations but not abolished. We show that the residual expression of IF2 beta results from translational initiation on an AUG codon located 21 bases downstream from the mutated GUG. Furthermore, two forms of IF2 beta have been separated by fast protein liquid chromatography and the determination of their N-terminal sequences indicated that they resulted from two internal initiation events, one occurring on the previously identified GUG start codon, the other on the AUG codon immediately downstream. We conclude that two forms of IF2 beta exist in the cell, which differ by seven aminoacid residues at their N terminus. Only by mutating both IF2 beta start codons could we construct plasmids that express only IF2 alpha. A plasmid expressing only IF2 beta was obtained by deletion of the proximal region of the infB gene. Using a strain that carries a null mutation in the chromosomal copy of infB and a functional copy of the same gene on a thermosensitive lysogenic lambda phage, we could cure the lambda phage when the plasmids expressing only one form of IF2 were supplied in trans. We found that each one of the two forms of IF2, at near physiological levels, can support growth of Escherichia coli, but that growth is retarded at 37 degrees C. This result shows that both forms of IF2 are required for maximal growth of the cell and suggests that they have acquired some specialized but not essential function.
Sacerdot
C
C
Institut de Biologie de Physico-Chimique, URA CNRS 1139, Paris, France.
Vachon
G
G
Laalami
S
S
Morel-Deville
F
F
Cenatiempo
Y
Y
Grunberg-Manago
M
M
D000595:Amino Acid Sequence; D001483:Base Sequence; D015153:Blotting, Western; D003001:Cloning, Molecular; D003062:Codon; D004269:DNA, Bacterial; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics / Q000254:growth & development*; D015852:Eukaryotic Initiation Factor-2 / Q000235:genetics / Q000502:physiology; D005816:Genetic Complementation Test; D008969:Molecular Sequence Data; D016296:Mutagenesis; D012329:RNA, Bacterial
D003062:Codon; D004269:DNA, Bacterial; D015852:Eukaryotic Initiation Factor-2; D012329:RNA, Bacterial