Era GTPase of Escherichia coli: binding to 16S rRNA and modulation of GTPase activity by RNA and carbohydrates

摘要

Era, an essential GTPase, appears to play an important role in the regulation of the cell cycle and protein synthesis of bacteria and mycoplasmas. In this study, native Era, His-tagged Era (His–Era) and glutathione S-transferase (GST)-fusion Era (GST–Era) proteins from Escherichia coli were expressed and purified. It was shown that the GST–Era and His–Era proteins purified by 1-step affinity column chromatographic methods were associated with RNA and exhibited a higher GTPase activity. However, the native Era protein purified by a 3-step column chromatographic method had a much lower GTPase activity and was not associated with RNA which had been removed during purification. Purified GST–Era protein was shown to be present as a high- and a low-molecular-mass forms. The high-molecular-mass form of GST–Era was associated with RNA and exhibited a much higher GTPase activity. Removal of the RNA associated with GST–Era resulted in a significant reduction in the GTPase activity. The RNA associated with GST–Era was shown to be primarily 16S rRNA. A purified native Era protein preparation, when mixed with total cellular RNA, was found to bind to some of the RNA. The native Era protein isolated directly from the cells of a wild-type E. coli strain was also present as a high-molecular-mass form complexed with RNA and RNase treatment converted the high-molecular-mass form into a 32?kDa low-molecular-mass form, a monomer of Era. Furthermore, a C-terminally truncated Era protein, when expressed in E. coli, did not bind RNA. Finally, the GTPase activity of the Era protein free of RNA, but not the Era protein associated with the RNA, was stimulated by acetate and3-phosphoglycerate. These carbohydrates, however, failed to activate the GTPase activity of the C-terminally truncated Era protein. Thus, the results of this study establish that the C-terminus of Era is essential for the RNA-binding activity and that the RNA and carbohydrates modulate the GTPase activity of Era possibly through a similar mechanism.