THE HINGE REGION OF ESCHERICHIA-COLI RIBOSOMAL-PROTEIN L7 L12 IS REQUIRED FOR FACTOR-BINDING AND GTP HYDROLYSIS/

A variant form of Escherichia coli ribosomal protein L7/L12 that lacke d residues 42 to 52 (L7/L12:Delta 42-52) in the hinge region was shown previously to be completely inactive in supporting polyphenylalanine synthesis although it bound to L7/L12 deficient core particles with th e normal stoichiometry of four copies per particle (Oleinikov AV, Perr oud B, Wang B, Traut RR (1993) J Biol Chem, 268, 917-922). The result suggested that the hinge confers flexibility that is required for acti vity because the resulting bent conformation allows the distal C-termi nal domain to occupy a location on the body of the large ribosomal sub unit proximal to the base of the L7/L12 stalk where elongation factors bind. Factor binding to the hinge-truncated variant was tested. As an alternative strategy to deleting residues from the hinge, seven amino acid residues within the putative hinge region were replaced by seven consecutive proline residues in an attempt to confer increased rigidi ty that might reduce or eliminate the bending of the molecule inferred to be functionally important. This variant, L7/L12:(Pro)(7), remained fully active in protein synthesis. Whereas the binding of both factor s in ribosomes containing L7/L12:Delta 42-52 was decreased by about 50 %, there was no loss of factor binding in ribosomes containing L7/L12: (Pro)(7), as predicted from the retention of protein synthesis activit y. The factor:ribosome complexes that contained L7/L12:Delta 42-52 had the same low level of GTP hydrolysis as the core particles completely lacking L7/L12 and EF-G did not support translocation measured by the reaction of phe-tRNA bound in the A site with puromycin. It is conclu ded that the hinge region is required for the functionally productive binding of elongation factors, and the defect in protein synthesis rep orted previously is due to this defect. The variant produced by the in troduction of the putative rigid Pro(7) sequence retains sufficient fl exibility for full activity.