A HYDROPHOBIC SEGMENT WITHIN THE 81-AMINO-ACID DOMAIN OF TFIIIA FROM SACCHAROMYCES-CEREVISIAE IS ESSENTIAL FOR ITS TRANSCRIPTION FACTOR ACTIVITY

Transcription factor IIIA (TFIIIA) binds to the internal control regio n of the 5S RNA gene as the first step in the in vitro assembly of a T FIIIB-TFIIIC-TFIIIA-DNA transcription complex, An 81-amino-acid domain that is present between zinc fingers 8 and 9 of TFIIIA from Saccharom yces cerevisiae is essential for the transcription factor activity of this protein (C. A. Milne and J. Segall, J. Biol. Chem. 268:11364-1137 1, 1993). We have monitored the effect of mutations within this domain on the ability of TFIIIA to support transcription of the 5S RNA gene in vitro and to maintain cell viability, TFIIIA with internal deletion s that removed residues 282 to 315, 316 to 334, 328 to 341, or 342 to 351 of the 81-amino-acid domain retained activity, whereas TFIIIA with a deletion of the short leucine-rich segment (352)NGLNLLLN(359) at th e carboxyl-terminal end of this domain was devoid of activity, Analysi s of the effects of double and quadruple mutations in the region exten ding from residue 336 to 364 confirmed that hydrophobic residues in th is portion of the 81-amino-acid domain, particularly L343, L347, L354, L356, L357, and L358, and to a lesser extent F336 and L337, contribut ed to the ability of TFIIIA to promote transcription. We propose that these hydrophobic residues play a role in mediating an interaction bet ween TFIIIA and another component of the transcriptional machinery, We also found that TFIIIA remained active if either zinc finger 8 or zin c finger 9 was disrupted by mutation but that TFIIIA containing a disr uption of both zinc finger 8 and zinc finger 9 was inactive.