The influence of transcription factor (TF) IIE on mRNA synthesis in vivo was examined in a temperature-sensitive yeast mutant. A missense mutation in the conserved zinc finger domain severely weakened TFIIE's transcription activity without appreciably affecting its quaternary structure, chromatographic properties, or cellular abundance. The mutation conferred recessive slow-growth and heat-sensitive phenotypes in yeast, but quantitative effects on promoter utilization by RNA polymerase II ranged from strongly negative to somewhat positive. Heat-induced activation of the HSP26, HSP104, and SSA4 genes was attenuated in the mutant, indicating dependence on TFIIE for maximal rates of de novo synthesis. Constitutive HSP expression in mutant cells was elevated, exposing a negative (likely indirect) influence by TFIIE in the absence of heat stress. Our results corroborate and extend recent findings of differential dependence on TFIIE activity for yeast promoters, but reveal an important counterpoint to the notion that dependence is tied to TATA element structure (Sakurai, H., Ohishi, T., and Fukasawa, T. (1997) J. Biol. Chem. 272, 15936-15942). We also provide empirical evidence for conservation of structure-activity relationships in TFIIE's zinc finger domain, and establish a direct link between TFIIE's biochemical activity in reconstituted transcription and its function in cellular mRNA synthesis.