Structural And Functional Study Of Ribosome Binding Protein ObgE

GTPases are molecular switches that regulate a variety of cellular events in biological systems by oscillating between two conformational states, GTP or GDP-bound state. Obg proteins are a family of P-loop GTPases, conserved from bacteria to human. Obg proteins have been implicated in many diverse cellular functions, for example in E. coli, Obg E is proposed to participate in two global processes, ribosome assembly and stringent response. But none of these above functions is understood at the molecular level.In the present work, we employed structural and bioc hemical approaches to study the structure and function of Obg E in E.coli.First of all, using cryo-electron microscopy(cryo-EM), we study the structure of the 50 S · Obg E · GMPPNP complex, which indicates that the evolutionarily conserved N-terminal domain of Obg E is a t RNA structural mimic, with specific interactions with peptidyl-transferase center(PTC), displaying a marked resemblance to Class I release factors. Besides, the association of Obg E brings large conformational changes to the 50 S subunit, which makes it unlikely for the small and large subunits to form 70 S ribosome. Along these lines, we use pre-steady state fast kinetics to demonstrate that Obg E is indeed an anti-association factor, which prevents ribosomal subunit association and downstream steps in translation by binding to the 50 S subunit. Moreover, Obg E is a ribosome dependent GTPase, however, upon binding to guanosine tetraphosphate(pp Gpp), the global regulator of stringent response, Obg E exhibits an enhanced interaction with the 50 S subuni t, resulting in increased equilibrium dissociation of the 70 S ribosome into subunits. Our data thus define Obg E as a specialized translation factor related to stress responses, and provide a framework towards future elucidation of functional interplay between Obg E and ribosome-associated(p)pp Gpp regulators.Together with published data, we thus propose that Obg E is a checkpoint protein in the assembly of 50 S subunit, which senses the c ellular energy stress via the level of(p)pp Gpp and links ribosome assembly to other global growth control pathways.