COTRANSLATIONAL FOLDING OF NASCENT PROTEINS ON ESCHERICHIA-COLI RIBOSOMES

Evidence is presented for cotranslational folding of rhodanese or rici n during its synthesis on Escherichia coli ribosomes. During transcrip tion-translation, full-length but enzymatically inactive polypeptides accumulated as peptidyl-tRNA on the ribosomes. These polypeptides were activated and released by subsequent incubation with the bacterial ch aperones and with release factor (RF-2). Coumarin was incorporated cot ranslationally at the N-terminus of the nascent protein from fluoropho re-S-Ac-Met-tRNA(f) Changes in fluorescence indicated that DnaJ bound to the nascent proteins and to a fluorescently labeled synthetic pepti de corresponding to the N-terminal 17 amino acids of bovine rhodanese. This peptide also bound to 70S ribosomes or 50S subunits but not to 3 0S subunits. It inhibited activation and RF-2-dependent release of the full-length ribosome-bound rhodanese. A deletion mutant of rhodanese lacking the N-terminal 23 amino acids was not accumulated on the ribos ome but was synthesized very efficiently. However, the protein that wa s formed was enzymatically inactive. DnaJ did not bind to this deletio n mutant on ribosomes. We conclude that the chaperone-mediated reactio ns facilitate binding of the N-terminal sequence of nascent proteins t o a specific site on 50S ribosomal subunits where it blocks release. T he ribosome-bound protein undergoes chaperone-mediated reactions that are required for folding into an enzymatically active conformation.