Abstract
Two lines of evidence indicate the importance of the N-terminal portion of rhodanese for correct folding of the nascent ribosome-bound polypeptide. A mutant gene lacking the codons for amino acids 1-23 of the wild-type protein is expressed very efficiently by coupled transcription/translation on Escherichia coli ribosomes; however, the mutant protein that is released from the ribosomes is enzymatically inactive. The mutant protein does not undergo the reaction that is promoted by the bacterial 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 incorporated at the N terminus of nascent rhodanese. Secondly, a synthetic peptide corresponding to the N-terminal 17 amino acids of the wild-type protein interferes with the synthesis of wild-type rhodanese but has much less effect on the synthesis of the N-terminal deletion mutant. The N-terminal peptide inhibits the effect of DnaJ on the nascent wild-type rhodanese and blocks the chaperone-mediated release and activation of ribosome-bound full-length rhodanese polypeptides that accumulate during in vitro synthesis. The results lead to the hypothesis that the N-terminal segment of rhodanese is required for its chaperone-dependent folding on the ribosome.
Highlights
Two lines of evidence indicate the importance of the N-terminal portion of rhodanese for correct folding of the nascent ribosome-bound polypeptide
A mutant gene lacking the codons for amino acids 1-23 of the wild-type protein is expressed very efficiently by coupled transcription/translation on Escherichia coli ribosomes; the mutant protein that is released from the ribosomes is enzymatically inactive
The mutant protein does not undergo the reaction that is promoted by the bacterial chaperone, DnaJ, which appears to be essential for folding of ribosome-bound rhodanese into the native conformation
Summary
Two lines of evidence indicate the importance of the N-terminal portion of rhodanese for correct folding of the nascent ribosome-bound polypeptide. The mutant protein does not undergo the reaction that is promoted by the bacterial chaperone, DnaJ, which appears to be essential for folding of ribosome-bound rhodanese into the native conformation. The N-terminal peptide inhibits the effect of DnaJ on the nascent wild-type rhodanese and blocks the chaperone-mediated release and activation of ribosome-bound full-length rhodanese polypeptides that accumulate during in vitro synthesis. As a complex with the nascent protein These studies indicate that at least some of the chaperone-mediated reactions by which nascent proteins are folded into their native conformation occur on ribosomes. The consensus features for targeting to the mitochondria are apparent in the N-terminal 31 amino acids of the native enzyme This peptide has seven positive charges that are embedded in what otherwise is a relatively hydrophobic sequence. The crystal structure of the protein shows that residues 11-22 form an a-helix and that the N terminus is positioned on the surface of one of the two globular domains (Ploegman et al, 1978; HoI et al, 1983)
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