Isolation and Characterization of a Dominant Negative Mutant of Bacillus subtilis GTP-binding Protein, YlqF, Essential for Biogenesis and Maintenance of the 50 S Ribosomal Subunit

• Published in: The Journal of biological chemistry 2007-08, Vol.282 (35), p.25270-25277
• Main Authors: Matsuo, Yoshitaka, Oshima, Taku, Loh, Pek Chin, Morimoto, Takuya, Ogasawara, Naotake
• Format: Article
• Language: English
• Subjects: Bacillus subtilis; Bacillus subtilis - genetics; Bacillus subtilis - metabolism; Bacterial Proteins - genetics; Bacterial Proteins - metabolism; Genes, Bacterial; Genes, Dominant; GTP-Binding Proteins - genetics; GTP-Binding Proteins - metabolism; Mutation; Nucleic Acid Conformation; Ribosomal Proteins - genetics; Ribosomal Proteins - metabolism; Ribosomes - genetics; Ribosomes - metabolism; RNA, Bacterial - genetics; RNA, Bacterial - metabolism; RNA, Ribosomal, 23S - genetics; RNA, Ribosomal, 23S - metabolism
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M703894200

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 (YlqFΔN10) 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, YlqFΔN10 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 YlqFΔN10-overexpressing cells and in YlqF-depleted cells, suggesting that YlqFΔN10 binds to the premature 50 S subunit. Moreover, premature 50 S subunit from both YlqFΔN10-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.