THE IMPORTANCE OF THE N-TERMINAL SEGMENT FOR DNAJ-MEDIATED FOLDING OFRHODANESE WHILE BOUND TO RIBOSOMES AS PEPTIDYL-TRANSFER-RNA

Two lines of evidence indicate the importance of the N-terminal portio n of rhodanese for correct folding of the nascent ribosome-bound polyp eptide. A mutant gene lacking the codons for amino acids 1-23 of the w ild-type protein is expressed very efficiently by coupled transcriptio n/translation on Escherichia coil ribosomes; however, the mutant prote in that is released from the ribosomes is enzymatically inactive. The mutant protein does not undergo the reaction that is promoted by the b acterial chaperone, DnaJ, which appears to be essential for folding of ribosome-bound rhodanese into the native conformation, The effect of DnaJ is monitored by fluorescence from coumarin cotranslationally inco rporated at the N terminus of nascent rhodanese. Secondly, a synthetic peptide corresponding to the N-terminal 17 amino acids of the wild-ty pe protein interferes with the synthesis of wild-type rhodanese but ha s much less effect on the synthesis of the N-terminal deletion mutant. The N-terminal peptide inhibits the effect of DnaJ on the nascent wil d-type rhodanese and blocks the chaperone-mediated release and activat ion of ribosome-bound full-length rhodanese polypeptides that accumula te during in, vitro synthesis. The results lead to the hypothesis that the N-terminal segment of rhodanese is required for its chaperone-dep endent folding on the ribosome.