Molecular cloning and sequence of the Bacillus stearothermophilus translational initiation factor IF2 gene.

Abstract

The structural gene for the Bacillus stearothermophilus initiation factor IF2 was localized to a 6 kb HindIII restriction fragment by cross-hybridization with the SstI-SmaI fragment of the Escherichia coli infB gene. This fragment corresponds to the central region of the molecule containing the GTP-binding domain which is homologous in E. coli IF2, EF-Tu, EF-G and the human ras1 oncogene protein. After cloning into pACYC177, the HindIII fragment was further analysed by restriction mapping and cross-hybridization. A smaller (2.2 kb) SphI-HindIII fragment, which showed cross-hybridization, was subcloned into M13 phage and sequenced by the dideoxy chain-terminating method. This fragment was found to contain the entire IF2 gene except for the region coding for the N-terminus. This remaining region, coding for 45 amino acids, was located by homologous hybridization on an overlapping ClaI-SstI fragment which was also subcloned and sequenced. Overall, the B. stearothermophilus IF2 gene codes for a protein of 742 amino acids (Mr = 82,043) whose primary sequence displays extensive homology with the C-terminal two-thirds (but little or no homology with the N-terminal one-third) of the corresponding E. coli IF2 molecule. When cloned into an expression vector under the control of the lambda PL promoter, the B. stearothermophilus IF2 gene, reconstituted by ligation of the two separately cloned pieces, could be expressed at high levels in E. coli cells.