MAPPING REGIONS OF YEAST TRANSCRIPTION FACTOR-IIIA REQUIRED FOR DNA-BINDING, INTERACTION WITH TRANSCRIPTION FACTOR-IIIC, AND TRANSCRIPTION ACTIVITY

TFIIIA, a site-specific DNA-binding protein containing nine zinc finge r motifs, is an RNA polymerase III transcription factor responsible fo r the assembly of a transcription complex on the 5 S RNA gene. We have analyzed the effect of deleting various regions of yeast TFIIIA on it s ability to bind to the internal control region of the yeast 5 S RNA gene, to recruit TFIIIC into the TFIIIA.DNA complex and to promote in vitro transcription of the 5 S RNA gene. A truncated polypeptide conta ining only the three amino-terminal zinc fingers retained the ability to bind specifically to the internal control region of the 5 S RNA gen e. The TFIIIA.DNA complex formed with this three zinc finger module wa s able to recruit TFIIIC but could not promote transcription. An 81-am ino acid domain, which interrupts the repeating zinc finger motifs bet ween fingers 8 and 9, was found to be essential for the transcription activity of the protein. Removal of the carboxyl-terminal region of ye ast TFIIIA that extends beyond the ninth zinc finger motif had no effe ct on the in vitro activities of the protein. Extending the carboxyl-t erminal deletion to include the ninth zinc finger led to reduced trans cription activity. We speculate that the ninth zinc finger serves to a nchor the carboxyl-terminal portion of TFIIIA on the 5 S RNA gene, cor rectly orienting the 81-amino acid domain to serve its role in promoti ng transcription. Removal of the region of the protein amino-terminal to the first zinc finger motif was without effect. Extending the amino -terminal deletion to include the first zinc finger appeared to decrea se the affinity of TFIIIA for DNA without affecting the specificity of the protein-DNA interaction. The resultant TFIIIA.DNA complex was def ective in recruiting TFIIIC and did not support transcription.