Abstract 2056 DEAD-box RNA helicase DbpA and RNA chaperone Hfq molecular functions are interconnected during ribosome biogenesis in Escherichia coli

The DbpA protein is a DEAD-box RNA helicase involved in the Peptidyl Transferases Center (PTC) maturation in Escherichia coli (E. coli) and its function is conserved in human PTC maturation. Expression in E. coli cells of the helicase inactive R331A DbpA construct produces the accumulation of three large subunit ribosome intermediates with sedimentation coefficients of 27S, 35S and 45S. We investigated by mass spectrometry the protein compositions of the large subunit intermediates accumulated in cells expressing R331A DbpA. We found the RNA chaperon Hfq present in the 27S and 35S intermediates. Very recently, it was shown that Hfq is involved in large subunit ribosome assembly in E. coli. In this work, using genetics and cell growth experiments, we investigated the interconnection between DbpA and Hfq cellular functions. The cells that lacked the dbpA gene showed no growth phenotype in rich media at different temperatures. On the other hand, the cells lacking the hfq gene showed a slow growth phenotype in rich media at all the temperatures investigated. Lastly, the cells that lacked both the dbpA and hfq genes showed a more severe growth defect in rich media than the cells lacking only the hfq gene. Interestingly, in minimal media at 25°C, the removal of the hfq gene is advantageous for the cells. Conversely, in minimal media at 25°C, the removal of the dbpA gene produces a slow growth phenotype. Lastly, in minimal media at 25°C, the cells lacking both genes grow similarly to wild type cells. Together, our genetic and growth data demonstrate that the functions of the DbpA and Hfq proteins are interconnected. Future experiments will decipher the molecular details of the DbpA and Hfq function interconnection during large subunit ribosome maturation. This work was supported in part by the National Institute of General Medical Sciences grant R01-GM131062, the University of Texas System Rising STARs Program, and the start-up from the Chemistry and Biochemistry Department at the University of Texas at El Paso (to E.K.).