Chaperone-dependent folding and activation of ribosome-bound nascent rhodanese. Analysis by fluorescence.
Fluorescently labeled rhodanese was synthesized by coupled transcription/translation in a cell-free Escherichia coli system. A derivative of coumarin was co-translationally incorporated at the N terminus of the polypeptide. Molecules released from the ribosomes during the incubation are enzymatically active; however, continued incubation results in accumulation of enzymatically inactive full-length rhodanese polypeptides on the ribosomes. These can be activated and released in the presence of the added chaperones, DnaJ, DnaK, GrpE, GroEL, GroES and ATP. Fluorescence parameters (quantum yield, anisotropy and the emission maximum) of ribosome-bound coumarin-labeled rhodanese are affected differentially by addition of the chaperones individually or sequentially. Rhodanese released from the ribosomes in the presence of all chaperones (enzymatically active) differs in fluorescence properties from rhodanese released by GroES or DnaK only or by puromycin (enzymatically inactive) indicating a difference in conformation. Using sparsomycin, an inhibitor of the peptidyl transferase reaction, full-length rhodanese can be trapped on the ribosomes. A ribosome-bound intermediate formed by DnaJ or DnaJ plus DnaK was demonstrated by the effect of these chaperones on fluorescence spectra resulting from binding of anticoumarin antibodies to the N terminus of newly synthesized rhodanese. The results support the hypothesis that folding of nascent proteins can take place on the ribosome.
University of Texas at Austin 78712.
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Kudlicki
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Odom
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Kramer
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Hardesty
D018833:Chaperonins / Q000378:metabolism*; D003374:Coumarins; D004926:Escherichia coli; D007074:Immunoglobulin G; D017510:Protein Folding; D012270:Ribosomes / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D013884:Thiosulfate Sulfurtransferase / Q000378:metabolism*
D003374:Coumarins; D007074:Immunoglobulin G; C030123:coumarin; D013884:Thiosulfate Sulfurtransferase; D018833:Chaperonins