Cooperative and critical roles for both G domains in the GTPase activity and cellular function of ribosome-associated Escherichia coli EngA.

To probe the cellular phenotype and biochemical function associated with the G domains of Escherichia coli EngA (YfgK, Der), mutations were created in the phosphate binding loop of each. Neither an S16A nor an S217A variant of G domain 1 or 2, respectively, was able to support growth of an engA conditional null. Polysome profiles of EngA-depleted cells were significantly altered, and His(6)-EngA was found to cofractionate with the 50S ribosomal subunit. The variants were unable to complement the abnormal polysome profile and were furthermore significantly impacted with respect to in vitro GTPase activity. Together, these observations suggest that the G domains have a cooperative function in ribosome stability and/or biogenesis.