Kudlicki W, Odom O W, Kramer G, Hardesty B
Department of Chemistry & Biochemistry, University of Texas, Austin 78712.
J Biol Chem. 1994 Jun 17;269(24):16549-53.
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.
在源自大肠杆菌的无细胞偶联转录/翻译系统中合成硫氰酸酶，会导致全长硫氰酸酶蛋白在核糖体上积累，同时也会产生从核糖体释放到上清液部分的具有酶活性的蛋白。核糖体结合的蛋白没有酶活性，但在添加伴侣蛋白DnaJ、DnaK、GrpE、GroEL和GroES以及ATP的情况下进行后续孵育，无需额外的蛋白质合成，就可以被激活并从核糖体上释放出来。高效激活要求在孵育过程中所有伴侣蛋白同时存在，这样才能产生完全有活性的硫氰酸酶。仅在DnaJ存在下孵育会抑制释放，而仅与GroES或DnaK孵育则会促进无酶活性蛋白的释放。用嘌呤霉素孵育核糖体可导致无酶活性蛋白的释放，而在所有伴侣蛋白存在下的释放和激活则会被稀疏霉素阻断。这些抗生素的作用提供了非常有力的证据，表明无酶活性的全长硫氰酸酶以肽基 - tRNA的形式结合在核糖体上，并且其释放需要肽基转移酶反应。综合考虑，数据表明伴侣蛋白介导的硫氰酸酶折叠成有酶活性的天然构象的后期阶段与作为蛋白质合成反应周期一部分发生的终止和释放过程密切相关。
