16S rRNA is bound to era of Streptococcus pneumoniae

Era is an essential membrane-associated GTPase that is present in bacteria and mycoplasmas. Era appears to play an important role in the regulation of the bacterial cell cycle. In this study, we expressed the native and gluta thione S-transferase (GST) fusion forms of Streptococcus pneumoniae Era in Escherichia coli and purified both proteins to homogeneity. We showed that RNA was copurified with the GST-Era protein of S. pneumoniae during affinit y purification and remained associated with the protein after removal of th e GST tag by thrombin cleavage. The thrombin-treated and untreated GST-Era proteins could bind and hydrolyze GTP and exhibited similar kinetic propert ies (dissociation constant [k(D)], K-m, and V-max). However, the native Era protein purified by using different chromatographic columns had a much low er GTPase activity than did GST-Era, although it had a similar k(D). In add ition, RNA was not associated with the protein. Purified GST-Era protein wa s shown to be present as high (600-kDa)- and low (120-kDa)-molecular-mass f orms. The high molecular-mass form of GST-Era was associated with RNA and e xhibited a very high GTPase activity. Approximately 40% of purified GST-Era protein was associated with RNA, and removal of the RNA resulted in a sign ificant reduction in GTPase activity. The RNA associated with GST-Era was s hown to be predominantly 16S rRNA. The native Era protein isolated directly from S. pneumoniae was also present as a high-molecular-mass species (600 kDa) complexed with RNA. Together, our results suggest that 16S rRNA is ass ociated with Era and might stimulate its GTPase activity.