FOLDING OF FIREFLY LUCIFERASE DURING TRANSLATION IN A CELL-FREE SYSTEM

In vitro synthesis of firefly luciferase and its folding into an enzym atically active conformation were studied in a wheat germ cell-free tr anslation system. A novel method is described by which the enzymatic a ctivity of newly synthesized luciferase can be monitored continuously in the cell-free system while this protein is being translated from it s mRNA. It is shown that ribosome-bound polypeptide chains have no det ectable enzymatic activity, but that this activity appears within a fe w seconds after luciferase has been released from the ribosome. In con trast, the renaturation of denatured luciferase under identical condit ions occurs with a half-time of 14 min. These results support the cotr anslational folding hypothesis which states that the nascent peptides start to attain their native tertiary structure during protein synthes is on the ribosome.