Functional analysis of the Bacillus subtilis GTP-binding protein family

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GTP-binding proteins are highly conserved and play critical roles in various cellular processes. The Bacillus subtilis genome encodes 7 essential GTP binding proteins, Era, Obg, YphC, YsxC, YlqF, YqeH, and YloQ, belonging to the Obg/Era subfamily of small GTP binding proteins, which had been poorly characterized. We demonstrated that YlqF is co-fractionated with the 50S ribosome subunit, in the presence of noncleavable GTP analog. Moreover, the GTPase activity of YlqF was stimulated specifically by the 50S subunit in vitro. Dimethyl sulfate footprinting analysis disclosed that YlqF is positioned around the A-site and P-site on the 50S subunit. Next, we found that an N-terminal deletion mutant of YlqF (YlqF_N10) inhibits cell growth even in the presence of wild-type YlqF. The GTPase activity of this mutant was not stimulated by the 50S subunit and did not dissociate from the premature 50S subunit. Premature 50S subunit accumulated in YlqF_N10-overexpressing cells and in YlqF depleted cells, suggesting that YlqF is targeted to the premature 50S subunit lacking ribosomal proteins L16 and L27 to assemble functional 50S subunit through a GTPase activity-dependent conformational change of 23S rRNA. Furthermore, we investigated functional relationships among Obg, YsxC, YphC and YlqF, and proposed a model for their sequential action in the late step of 50S subunit biogenesis. In contrast, Era, YloQ and YqeH are involved in the 30S subunit biogenesis. We demonstrated a reduction in the 70S ribosome and accumulation of the free 50S subunit in YqeH-depleted cells and our results strongly suggested that YqeH and Era function at distinct checkpoints during 30S subunit assembly. In addition, we have found that that alternative localization of two types of ribosomal proteins in the B. subtilis ribosome depending on the intracellular concentration of zinc, zinc-binding motif containing type (RpmE or RpsN) and their homologues without the motif (YtiA or YhzA).