Tandem translation of Bacillus subtilis initiation factor IF2 in E. coli. Over-expression of infBB.su in E. coli and purification of alpha- and beta-forms of IF2B.su.
The protein synthesis initiation factor, IF2, in Bacillus subtilis has previously been characterized as being present in two forms, alpha and beta, of molecular mass 79 and 68 kDa, respectively, on the basis of their cross-reaction with anti-E. coli IF2 antibodies and by the DNA sequence of the gene for IF2, infBB.su. In this work we have cloned infBB.su in E. coli cells. Two proteins of molecular mass identical to the B. subtilis IF2 alpha and -beta were over-expressed and purified using a new three-step ion-exchange chromatography procedure. The N-terminal amino acid sequence of the two proteins was determined and the results confirmed that the two forms were IF2 alpha and -beta, both encoded by the infB gene. The N-terminal amino acid sequence determined for IF2 beta is Met94-Gln-Asn-Asn-Gln-Phe. The presence of methionine at position 94 shows that this form is, in fact, the result of a second translational initiation in infBB.su mRNA, since the codon at amino acid position 94 is GUG, which is the normal codon for valine, but also known to be an initiator codon. This is a new example of the unusual tandem translation in E. coli of an open mRNA reading frame.
Hubert
M
M
Department of Chemistry, Aarhus University, Denmark.
Nyengaard
N R
NR
Shazand
K
K
Mortensen
K K
KK
Lassen
S F
SF
Grunberg-Manago
M
M
Sperling-Petersen
H U
HU
D000595:Amino Acid Sequence; D001412:Bacillus subtilis / Q000235:genetics*; D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism; D001483:Base Sequence; D002850:Chromatography, Gel; D003001:Cloning, Molecular; D004269:DNA, Bacterial; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics*; D008969:Molecular Sequence Data; D016366:Open Reading Frames; D010448:Peptide Initiation Factors / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D039881:Prokaryotic Initiation Factor-2; D014176:Protein Biosynthesis
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D010448:Peptide Initiation Factors; D039881:Prokaryotic Initiation Factor-2