A study has been carried out of the structural and functional domains of the Escherichia coli initiation factor IF2. An IF2 derived protein of molecular weight 55 kDa, or higher as long as it contained the C-terminal half supported growth of E coli and fulfilled all known functions of IF2. Cells expressing IF2 with the C-terminal quarter of amino acids deleted were not viable. Near the centre of IF2 there was a sequence conserved in proteins which binds guanosine triphosphate (GTP). A series of mutations was constructed in this binding site. The strain which survived with the short form of IF2 (55 kDa) in the absence of wild type IF2 allowed cloning and purification of these specific IF2 mutants based on the molecular weight difference (97.3 and 79.7 kDa). The activity of the mutants could then be studied in vitro in the absence of contaminating wild type initiation factor. These tools may be useful in molecular studies of the structure and activities of different domains of IF2 and the role of GTP in initiation of protein biosynthesis. A new procedure has also been developed for purification of large amounts of IF2a and IF2b. Further studies have characterized the Bacillus subtilis IF2 operon with a view to study of Bacillus subtilis infB regulation.
During the last 15-20 years the two laboratories involved have been among the leading research groups in the field of the molecular mechanisms of bacterial protein synthesis. Especial expertise has been build up on the structure and function of the macromolecules involved in the initiation step.
In the project presented, we wish to combine and to exchange many classical biochemical methods and recently-introduced methods of biotechnology in a close collaboration on the study of the relation between the structure and the function of a GTP-hydrolysing protein, initiation factor IF2 from Escherichia coli. The project includes the use and exchange of the newest methods of gene technology, immunoassays (preparation and characterisation of monoclonal antibodies), the purification of proteins and the determination of their structure. It has been found that a number of the chemical reactions in the living cell are regulated by GTP-hydrolysing proteins similar to IF2. The importance of the project is therefore not limited to the mechanism of initiation in bacterial protein synthesis, but can be expected to provide a much more general understanding of the function of GTP-hydrolising proteins.
Funding SchemeCSC - Cost-sharing contracts