The importance of the N-terminal segment for DnaJ-mediated folding of rhodanese while bound to ribosomes as peptidyl-tRNA.
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.
Kudlicki
W
W
Department of Chemistry and Biochemistry, University of Texas, Austin 78712, USA.
Odom
O W
OW
Kramer
G
G
Hardesty
B
B
Merrill
G A
GA
Horowitz
P M
PM
D029968:Escherichia coli Proteins; D050956:HSP40 Heat-Shock Proteins; D018840:HSP70 Heat-Shock Proteins / Q000378:metabolism; D006360:Heat-Shock Proteins / Q000378:metabolism*; D018832:Molecular Chaperones; D017510:Protein Folding; D012346:RNA, Transfer, Amino Acyl / Q000378:metabolism; D012270:Ribosomes / Q000378:metabolism*; D017384:Sequence Deletion; D013050:Spectrometry, Fluorescence; D013329:Structure-Activity Relationship; D013884:Thiosulfate Sulfurtransferase / Q000737:chemistry*
C494635:DnaJ protein, E coli; D029968:Escherichia coli Proteins; D050956:HSP40 Heat-Shock Proteins; D018840:HSP70 Heat-Shock Proteins; D006360:Heat-Shock Proteins; D018832:Molecular Chaperones; D012346:RNA, Transfer, Amino Acyl; C019037:tRNA, peptidyl-; D013884:Thiosulfate Sulfurtransferase; C063842:dnaK protein, E coli