Activation and release of enzymatically inactive, full-length rhodanese that is bound to ribosomes as peptidyl-tRNA.
Synthesis of rhodanese in a cell-free coupled transcription/translation system derived from Escherichia coli leads to an accumulation of full-length rhodanese protein on the ribosomes as well as to enzymatically active protein that is released from the ribosomes into the supernatant fraction. The ribosome-bound protein is enzymatically inactive but can be activated and released from the ribosomes without additional protein synthesis by subsequent incubation in the presence of the added chaperones DnaJ, DnaK, GrpE, GroEL, and GroES plus ATP. Efficient activation requires that all of the chaperones are present together during incubation which yields fully active rhodanese. Incubation in the presence of DnaJ only inhibits release whereas incubation with only GroES or DnaK promotes the release of enzymatically inactive protein. Incubation of the ribosome with puromycin leads to the release of enzymatically inactive protein whereas release and activation in the presence of all of the chaperones is blocked by sparsomycin. The effect of these antibiotics provides very strong evidence that enzymatically inactive, full-length rhodanese is bound to the ribosomes as peptidyl-tRNA and that the peptidyl transferase reaction is required for its release. Considered together, the data indicate that chaperone-mediated late stages of rhodanese folding into the enzymatically active, native conformation are intimately associated with the process of termination and release that occurs as part of the reaction cycle of protein synthesis.
Kudlicki
W
W
Department of Chemistry & Biochemistry, University of Texas, Austin 78712.
Odom
O W
OW
Kramer
G
G
Hardesty
B
B
D000818:Animals; D001426:Bacterial Proteins / Q000378:metabolism; D002417:Cattle; D002474:Cell-Free System; D004789:Enzyme Activation; D004926:Escherichia coli; D006360:Heat-Shock Proteins / Q000378:metabolism; D008930:Mitochondria, Liver / Q000201:enzymology; D011485:Protein Binding; D014176:Protein Biosynthesis; D012343:RNA, Transfer / Q000378:metabolism; D012346:RNA, Transfer, Amino Acyl / Q000378:metabolism*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D012270:Ribosomes / Q000378:metabolism*; D013884:Thiosulfate Sulfurtransferase / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D014158:Transcription, Genetic
D001426:Bacterial Proteins; D006360:Heat-Shock Proteins; D012346:RNA, Transfer, Amino Acyl; D011994:Recombinant Proteins; C019037:tRNA, peptidyl-; D012343:RNA, Transfer; D013884:Thiosulfate Sulfurtransferase