A Hydrophobic Segment within the 81-Amino-Acid Domain of TFIIIA from Saccharomyces cerevisiae Is Essential for Its Transcription Factor Activity

ABSTRACT

Transcription factor IIIA (TFIIIA) binds to the internal control region of the 5S RNA gene as the first step in the in vitro assembly of a TFIIIB-TFIIIC-TFIIIA-DNA transcription complex. An 81-amino-acid domain that is present between zinc fingers 8 and 9 of TFIIIA fromSaccharomyces cerevisiae is essential for the transcription factor activity of this protein (C. A. Milne and J. Segall, J. Biol. Chem. 268:11364–11371, 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 deletions 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 ³⁵²NGLNLLLN³⁵⁹ at the carboxyl-terminal end of this domain was devoid of activity. Analysis of the effects of double and quadruple mutations in the region extending from residue 336 to 364 confirmed that hydrophobic residues in this portion of the 81-amino-acid domain, particularly L343, L347, L354, L356, L357, and L358, and to a lesser extent F336 and L337, contributed to the ability of TFIIIA to promote transcription. We propose that these hydrophobic residues play a role in mediating an interaction between TFIIIA and another component of the transcriptional machinery. We also found that TFIIIA remained active if either zinc finger 8 or zinc finger 9 was disrupted by mutation but that TFIIIA containing a disruption of both zinc finger 8 and zinc finger 9 was inactive.

ACKNOWLEDGMENTS

We thank Catherine Milne for providing a yeast-derived heparin-agarose fraction h, Randall Willis for construction of the yeast strain YRW1, John Hwang for assistance in the construction of a number of the plasmids used in this study, and Shelley Hepworth for valuable comments regarding the manuscript.

O.R. was supported by an Ontario Graduate Scholarship. This work was supported by a Medical Research Council (Canada) grant (MA-6826) to J.S.