FIG. 1.

Reconstitution of protein priming with different combinations of chaperone proteins. Purified GST-MiniRT1 was used for an in vitro protein-priming assay, with or without reconstitution with the indicated chaperone proteins. All reactions were supplemented with the ɛ RNA and an ATP regenerating system (see Materials and Methods for details). Approximately 10 ng of GST-MiniRT1 was used in each reaction (10-μl reaction volume). The amount of the chaperone proteins used was 350 ng of Hsp70, 1,000 ng of Ydj1, 120 ng of Hsp90, 125 ng of Hop, and 20 ng of p23 per reaction. Reticulocyte lysate (RL) (Promega) was used at 5 μl per reaction. The reaction time in all cases was 1 h at 30°C. The ³²P-labeled GST-MiniRT1 protein (the product of protein priming) was detected by resolving the reactions by SDS-PAGE and autoradiography. The labeled GST-MiniRT1 protein is indicated (RT).