FIG. 5.

Effects of mutations that disrupt the structure of zinc fingers 8 and 9 on activity of TFIIIA. (A) Schematic representation of yeast TFIIIA as described for Fig. 1A. The approximate positions of mutations H272R and C367Y within zinc fingers 8 and 9, respectively, are indicated by arrows. (B) Abilities of versions of TFIIIA with disruptions of zinc fingers 8 and 9 to bind to the 5S RNA gene and to recruit TFIIIC to the TFIIIA-DNA complex, as outlined in the legend to Fig. 1C. Lanes 1 to 4 were as described for Fig. 1C. Lanes 5 to 10, in vitro-synthesized versions of TFIIIA containing the following mutation(s): H272R/C367Y (lanes 5 and 6), H272R (lanes 7 and 8), or C367Y (lanes 9 and 10). (C) Abilities of versions of TFIIIA with disruptions of zinc fingers 8 and 9 to support in vitro transcription of the 5S RNA gene. For details, see the legend to Fig. 1D. (D) Abilities of versions of TFIIIA with disruptions of zinc fingers 8 and 9 to support cell viability. A plasmid shuffle system was used to test the abilities of mutant versions of TFIIIA to support cell viability, as described in the legend to Fig. 2D. (E) Assessment by Western blot analysis of in vivo expression of versions of TFIIIA with disruptions in zinc fingers 8 and 9. The analysis was carried out as described in the legend to Fig. 2E. Protein was extracted from YRW1 cells containing pG3-derived plasmids expressing no TFIIIA (lane 1), wild-type TFIIIA (lane 2), TFIIIA(H272R/C367Y) (lane 3), TFIIIA(H272R) (lane 4), or TFIIIA(C367Y) (lane 5).