A single proteolytic cleavage in release factor 2 stabilizes ribosome binding and abolishes peptidyl-tRNA hydrolysis activity.
The structural and functional organization of Escherichia coli polypeptide chain release factors 1 and 2 (RF-1 and RF-2) was investigated by limited proteolysis with trypsin and chymotrypsin. A protease-sensitive site was found in a similar position in both factors at the beginning of a highly conserved region in the C-terminal part of the proteins. Chymotrypsin cleavage of RF-2 yielded a nicked form with the fragments associated. This nicked factor lost in vitro peptidyl-tRNA hydrolysis activity (a peptidyltransferase function) but had enhanced in vitro codon-ribosome binding activity (a decoding site function). It inhibited codon-dependent f[3H]Met-tRNA hydrolysis activity of intact RF-1 and RF-2, presumably as a result of an increased affinity for ribosomes. These data are consistent with a model whereby the release factor acts like a tRNA analog spanning the decoding and peptidyltransferase centers on the ribosome. The proteolytic sensitivity of the RFs most likely reflects an exposed surface loop. We propose that this loop interacts with the ribosomal peptidyltransferase site and that the stabilization of factor:ribosome binding upon cleavage could be explained by conformational coupling between domains on the factor for codon-ribosome binding at the decoding site and interaction with peptidyltransferase.
Department of Biochemistry, University of Otago, Dunedin, New Zealand.
J G
JG
Moffat
W P
WP
Tate
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000378:metabolism; D002918:Chymotrypsin / Q000494:pharmacology; D004926:Escherichia coli; D005656:Fungal Proteins / Q000378:metabolism; D008969:Molecular Sequence Data; D010443:Peptide Chain Termination, Translational; D010454:Peptide Termination Factors / Q000737:chemistry* / Q000378:metabolism; D010458:Peptidyl Transferases / Q000378:metabolism; D012346:RNA, Transfer, Amino Acyl / Q000378:metabolism*; D012270:Ribosomes / Q000378:metabolism*; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013329:Structure-Activity Relationship
D001426:Bacterial Proteins; D005656:Fungal Proteins; D010454:Peptide Termination Factors; D012346:RNA, Transfer, Amino Acyl; C041460:peptide chain termination release factor 2; C019037:tRNA, peptidyl-; D010458:Peptidyl Transferases; D002918:Chymotrypsin