W. Kudlicki et al., THE IMPORTANCE OF THE N-TERMINAL SEGMENT FOR DNAJ-MEDIATED FOLDING OFRHODANESE WHILE BOUND TO RIBOSOMES AS PEPTIDYL-TRANSFER-RNA, The Journal of biological chemistry, 270(18), 1995, pp. 10650-10657
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.