Isolation and molecular genetic characterization of the Bacillus subtilis gene (infB) encoding protein synthesis initiation factor 2

Abstract

Western blot (immunoblot) analysis of Bacillus subtilis cell extracts detected two proteins that cross-reacted with monospecific polyclonal antibody raised against Escherichia coli initiation factor 2a (IF2a). Subsequent Southern blot analysis of B. subtils genomic DNA identified a 1.3-kilobase (kb) Hindu fragment which cross-hybridized with both E. coli and Bacillus stearothermophilus IF2 gene probes. This DNA was cloned from a size-selected B. subtilis plasmid library. The cloned HindUI fragment, which was shown by DNA sequence analysis to encode the N-terminal half of the B. subtilis IF2 protein and 0.2 kb of upstream flanking sequence, was utilized as a homologous probe to clone an overlapping 2.76-kb ClaI chromosomal fragment containing the entire IF2 structural gene. The Hindm fragment was also used as a probe to obtain overlapping clones from a Agtll library which contained additional upstream and downstream flanking sequences. Sequence comparisons between the B. subtilis IF2 gene and the other bacterial homologs from E. coli, B. stearothermophilus, and Streptococcus faecium displayed extensive nucleic acid and protein sequence homologies. The B. subtilis injB gene encodes two proteins, IF2a (78.6 kilodaltons) and IF2( (68.2 kilodaltons); both were expressed in B.subtilis and E. coli. These two proteins cross-reacted with antiserum to E. coli IF2a and were able to complement in vivo an E. coli inJB gene disruption. Four-factor recombination analysis positioned the inJB gene at 145°on the B. subtilis chromosome, between the poiC and spcB loci.' This location is distinct from those of the other major ribosomal protein and rRNA gene clusters of B. subtilis.The initiation step of protein synthesis has been extensively studied in the gram-negative bacterium Escherichia coli by biochemical (15,17,23) and genetic (7, 16) techniques. Fractionation of E. coli cell extracts has resulted in the identification and purification of three protein synthesis initiation factors (IF1, IF2, and IF3 ) that stimulate the formation of the 30S preinitiation complex. The 30S'preinitiation complex is composed of a 30S ribosome subunit bound to initiation factors, mRNA, and 'formylmethionyl initiator tRNA (fMet-tRNA'et); a molecule of GTP is bound by IF2. IF2 appears to selectively discriminate in favor of fMet-tRNAfmet binding to the 30S subunit (21). IF3 is involved in additional aspects of the recognition and selection of fMet-tRNAfmt (21). IF3 also may participate in mRNA recognition (14). IF1 stimulates the activities of the other two initiation factors and is required for the selection of initiator tRNA by 70S complexes (21). IF1 and IF3 are released upon formation of a 70S ribosomal initiation complex. This coupling of ribosomal subunits, with the mRNA start codon base paired to the anticodon of fMet-tRNAfMet within the peptidyl site, is essentially irreversible. Hydrolysis of the IF2-bound GTP is apparently concomitant with the release of IF2 from the 70S initiation complex. Release of IF2 leaves a 70S ribosome poised to en...

Similar organization of the nusA-infB operon in Bacillus subtilis and Escherichia coli

“…3 encodes the last 40 amino acids of DNA polymerase III, the polC product. The presence of the polC gene in close proximity to infB is in good agreement with the previous determination of the infB chromosomal position (42 (Fig. 3).…”

“…1-Galactosidase assays were performed on sonically disrupted extracts as described before (44) (42) (Fig. 1).…”

“…Recent studies have identified homologs of infA in Bacillus subtilis (4), of infB in B. subtilis (42), Bacillus stearothermophilus (6), and Streptococcus faecium (10), and of infC in B. stearothennophilus (33). The Bacillus infA and infC genes have been shown to belong to operons encoding other ribosomal proteins (4,33).…”

“…Only part of the B. subtilis infB operon was cloned previously, and its structure, outside of the infB gene, was not characterized (42). The E. coli infB operon contains the metY gene encoding a minor form of initiator tRNA, three genes encoding proteins of unknown function (P15A, P15B, and P35K), infB itself, and the nusA gene encoding a protein which is involved in termination of transcription (15,20,32,39).…”

Macromolecular Mimicry in Translation Initiation: A Model for the Initiation Factor IF2 on the Ribosome

“…Thus, T. thermophilus IF2 may not require a speci c region of its structure with high af nity for the ribosome. However, T. thermophilus IF2 may not have a strong interaction with the ribosome compared to E. coli IF2 since Vornlocher and coworkers (35) found that during the purication of T. thermophilus IF2, the factor was almost completely Overall a high sequence similarity is found towards the C-terminus of the protein opposite to the higher variability around the N-terminus (52)(53)(54)(55).…”