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

Era, an essential GTPase, appears to play an important role in the regulati on of the cell cycle and protein synthesis of bacteria and mycoplasmas. In this study, native Era, His-tagged Era (His-Era) and glutathione S-transfer ase (GST)-fusion Era (CST-Era) proteins from Escherichia coli were expresse d 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 pu rified by a 3-step column chromatographic method had a much lower GTPase ac tivity and was not associated with RNA which had been removed during purifi cation. Purified GET-Era protein was shown to be present as a high- and a l ow-molecular-mass forms. The high-molecular-mass form of GET-Era was associ ated with RNA and exhibited a much higher GTPase activity. Removal of the R NA associated with GET-Era resulted in a significant reduction in the GTPas e activity. The RNA associated with GET-Era was shown to be primarily 16S r RNA. A purified native Era protein preparation, when mixed with total cellu lar RNA, was found to kind to some of the RNA. The native Era protein isola ted 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 conver ted the high-molecular-mass form into a 32 kDa low-molecular-mass form, a m onomer of Era. Furthermore, a C-terminally truncated Era protein, when expr essed 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 and 3-phosphoglycerate. These carbohydrates, however, failed to activate the GTPase activity of the C-terminally truncated Era p rotein. Thus, the results of this study establish that the C-terminus of Er a is essential for the RNA-binding activity and that the RNA and carbohydra tes modulate the GTPase activity of Era possibly through a similar mechanis m.