ACTIVATION AND RELEASE OF ENZYMATICALLY INACTIVE, FULL-LENGTH RHODANESE THAT IS BOUND TO RIBOSOMES AS PEPTIDYL-TRANSFER-RNA

Synthesis of rhodanese in a cell-free coupled transcription/translatio n system derived from Escherichia coli leads to an accumulation of ful l length rhodanese protein on the ribosomes as well as to enzymaticall y active protein that is released from the ribosomes into the supernat ant fraction. The ribosome-bound protein is enzymatically inactive but can be activated and released from the ribosomes without additional p rotein synthesis by subsequent incubation in the presence of the added chaperones DnaJ, DnaK, GrpE, GroEL, and GroES plus ATP. Efficient act ivation requires that all of the chaperones are present together durin g incubation which yields fully active rhodanese. Incubation in the pr esence of DnaJ only inhibits release whereas incubation with only GroE S or DnaK promotes the release of enzymatically inactive protein. Incu bation of the ribosome with puromycin leads to the release of enzymati cally inactive protein whereas release and activation in the presence of all of the chaperones is blocked by sparsomycin. The effect of thes e antibiotics provides very strong evidence that enzymatically inactiv e, full-length rhodanese is bound to the ribosomes as peptidyl-tRNA an d that the peptidyl transferase reaction is required for its release. Considered together, the data indicate that chaperone-mediated late st ages of rhodanese folding into the enzymatically active, native confor mation are intimately associated with the process of termination and r elease that occurs as part of the reaction cycle of protein synthesis.