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Bacillus subtilis YlqF belongs to the Era/Obg subfamily of small GTP-binding proteins and is essential for bacterial growth. Here we report that YlqF participates in the late step of 50 S ribosomal subunit assembly. YlqF was co-fractionated with the 50 S subunit, depending on the presence of noncleavable GTP analog. Moreover, the GTPase activity of YlqF was stimulated specifically by the 50 S subunit in vitro. Dimethyl sulfate footprinting analysis disclosed that YlqF binds to a unique position in 23 S rRNA. Yeast two-hybrid data revealed interactions between YlqF and the B. subtilis L25 protein (Ctc). The interaction was confirmed by the pull-down assay of the purified proteins. Specifically, YlqF is positioned around the A-site and P-site on the 50 S subunit. Proteome analysis of the abnormal 50 S subunits that accumulated in YlqF-depleted cells showed that L16 and L27 proteins, located near the YlqF-binding domain, are missing. Our results collectively indicate that YlqF will organize the late step of 50 S ribosomal subunit assembly.

Bacterial genome sequencing has revealed a novel family of P-loop GTPases that are often essential for growth. Accumulating evidence suggests that these proteins are involved in biogenesis of the 30S or 50S ribosomal subunits. YqeH is a member of this Obg/Era GTPase family, with its function remains to be uncovered. Here, we present results showing that YqeH is involved in the 30S subunit biogenesis in Bacillus subtilis. We observed a reduction in the 70S ribosome and accumulation of the free 50S subunit in YqeH-depleted cells. Interestingly, no free 30S subunit accumulation was evident. Consistent with the theory that YqeH is involved in 30S subunit biogenesis, a precursor of 16S rRNA and its degradation products were detected. Additionally, the reduction of free 30S subunit was not observed in Eradepleted cells. YqeH overexpression did not compensate for growth defects in mutants devoid of Era and vice versa. Moreover, in vitro GTPase analyses showed that YqeH possessed high intrinsic GTPase activity. In contrast, Era showed slow GTPase activity, which was enhanced by the 30S ribosomal subunit. Our findings strongly suggest that YqeH and Era function at distinct checkpoints during 30S subunit assembly. B. subtilis yqeH is classified as an essential gene due to the inability of the IPTG-dependent P spac -yqeH mutant to grow on LB or PAB agar plates in the absence of IPTG. However, in our experiments, the P spac -yqeH mutant grew in PAB liquid medium without IPTG supplementation, albeit at an impaired rate. This finding raises the interesting possibility that YqeH participates in assembly of the 30S ribosomal subunit as well as other cellular functions essential for growth on solid media.

The circularly permuted GTPase YlqF is essential for cell viability and is broadly conserved from Gram-positive bacteria to eukaryotes. We previously reported that YlqF participates in the late step of 50 S ribosomal subunit assembly in Bacillus subtilis. Here, we demonstrate that an N-terminal deletion mutant of YlqF (YlqF⌬N10) inhibits cell growth even in the presence of wild-type YlqF. In contrast to the wild-type protein, the GTPase activity of this mutant was not stimulated by the 50 S subunit and did not dissociate from the premature 50 S subunit. Thus, YlqF⌬N10 acts as a competitive inhibitor of wild-type YlqF. Premature 50 S subunit lacking ribosomal protein L27 and with a reduced amount of L16 accumulated in YlqF⌬N10-overexpressing cells and in YlqFdepleted cells, suggesting that YlqF⌬N10 binds to the premature 50 S subunit. Moreover, premature 50 S subunit from both YlqF⌬N10-overexpressing and YlqF-depleted cells more strongly enhanced the GTPase activity of YlqF than the mature 50 S subunit of the 70 S ribosome. Collectively, our results indicate that YlqF is targeted to the premature 50 S subunit lacking ribosomal proteins L16 and L27 to assemble functional 50 S subunit through a GTPase activity-dependent conformational change of 23 S rRNA.

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