Methionine Aminopeptidase Gene From Bacillus Subtilis Cloning And Functional Study

Ribosome-mediated protein biosynthesis is always initiated with either methionine (in eukaryotes) or N-formylmethionine (in prokaryotes and eukaryotic organelles). Methionine aminopeptidase (MAP) is responsible for removing N-terminal methionine from nascent peptides and the gene is essential to the cell. Each eukaryotic cell possesses two map gene copies while prokaryotes have only one copy in each genome, according to the papers so far. Bacillus subtilis, a Gram-positive bacterium whose whole genome sequence had just been finished in 1998, was chosen as the model system in this study. Sequence homology searching hinted there were two candidate genes for MAP function in B. subtilis, map and yflG respectively. Their proteins homology value is 65%. Both of these two genes were cloned into Escherichia coli for overexpression. Met-thioester-Phe, an analogue of tripeptide, as substrate, cobalt as cofactor, biochemical assay was tested, which showed that only Map is active while YflG has no enzymatic activity under this condition. E. coli P-(T7)-map mutant as host cell, map and yflG, whose expression was under the control of arabinose, were introduced in trans for genetic rescue study. It demonstrated both map and yflG can make the cell survive under the conditions of having arabinose but not IPTG, supporting they are functional in vivo in E. coli. Furthermore it has been shown successfully that in B. subtilis both of the genes code for MAP and their functions are replaceable. Lacking both of them is lethal to the cells. Besides characterisation of the two genes' function, RT-PCR, Northern analysis, primer extension, and lacZ reporter gene fusions were also tried in this study...