tailieunhanh - Báo cáo khoa học: Characterization of the 16S rRNA- and membrane-binding domains of Streptococcus pneumoniae Era GTPase Structural and functional implications

Era is a highly conserved GTPase essential for bacterial growth. The N-terminal part of Era contains a conserved GTPase domain, whereas the C-terminal part of the protein contains anRNA- andmembrane-binding domain, theKH domain. To investigate whether the binding of Era to 16S rRNA and membrane requires its GTPase activity and whether the GTPase domain is essential for these acti-vities, the N- and C-terminal parts of the Streptococ-cus pneumoniaeEra –Era-N(aminoacids 1–185) andEra-C (amino acids 141–299), respectively – were expressed and purified. . | Eur. J. Biochem. 270 4164-4172 2003 FEBS 2003 doi Characterization of the 16S rRNA- and membrane-binding domains of Streptococcus pneumoniae Era GTPase Structural and functional implications Julie Q. Hang1 and Genshi Zhao2 1 Roche Palo Alto LLC Palo Alto CA USA 2Cancer Research Lilly Research Laboratories Eli Lilly and Company Indianapolis IN USA Era is a highly conserved GTPase essential for bacterial growth. The N-terminal part of Era contains a conserved GTPase domain whereas the C-terminal part of the protein contains an RNA- and membrane-binding domain the KH domain. To investigate whether the binding of Era to 16S rRNA and membrane requires its GTPase activity and whether the GTPase domain is essential for these activities the N- and C-terminal parts of the Streptococcus pneumoniae Era - Era-N amino acids 1-185 and Era-C amino acids 141-299 respectively - were expressed and purified. Era-C which had completely lost GTPase activity bound to the cytoplasmic membrane and 16S rRNA. In contrast Era-N which retained GTPase activity failed to bind to RNA or membrane. These results therefore indicate that the binding of Era to RNA and membrane does not require the GTPase activity of the protein and that the RNA-binding domain is an independent functional domain. The physiological effects of the overexpression of Era-C were assessed. The Escherichia coli cells overexpress ing Era and Era-N exhibited the same growth rate as wildtype E. coli cells. In contrast the E. coli cells overexpressing Era-C exhibited a reduced growth rate indicating that the overexpression of Era-C inhibits cell growth. Furthermore overexpression of era-N and era-C resulted in morphological changes. Finally purified Era and Era-C were able to bind to poly U RNA and the binding of Era to poly U RNA was significantly inhibited by liposome as the amount of Era bound to the RNA decreased proportionally with the increase of liposome in the assay. Therefore this study