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 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 overexpressing Era and Era-N exhibited the same growth rate as wild-type 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 provides the first biochemical evidence that both binding sites are overlapping. Together, these results indicate that the RNA- and membrane-binding domain of Era is a separate, functional entity and does not require the GTPase activity or the GTPase domain of the protein for activity.
D001665:Binding Sites; D002455:Cell Division / Q000502:physiology; D029968:Escherichia coli Proteins; D019204:GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D008081:Liposomes / Q000378:metabolism; D011072:Poly U / Q000378:metabolism; D017434:Protein Structure, Tertiary; D012336:RNA, Ribosomal, 16S / Q000378:metabolism*; D016601:RNA-Binding Proteins; D013296:Streptococcus pneumoniae / Q000378:metabolism*
D029968:Escherichia coli Proteins; D008081:Liposomes; D012336:RNA, Ribosomal, 16S; D016601:RNA-Binding Proteins; C068027:era protein, E coli; D011072:Poly U; D019204:GTP-Binding Proteins