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The first unique step in the synthesis of the tobacco phytoalexin capsidiol is cyclization of farnesyl pyrophosphate catalyzed by 5-epi-aristolochene synthase (EAS), a sesquiterpene cyclase. Earlier work demonstrated that the elicitor-inducibility of this enzyme activity corresponded to the transcriptional activation of at least one gene, EAS4, of a rather complex gene family consisting of > 10 members. To investigate the mechanism(s) controlling expression of this gene, fragments of the EAS4 promoter were examined for binding by proteins in nuclear and whole-cell extracts. A strong protein binding site (TAC box; ACTCTACAGTACTC) was identified between −245 and −232 by the electrophoretic mobility shift assay, and DNAase I and methylation interference footprinting. Several distinctly migrating bands representing protein-TAC box complexes were also observed in the mobility shift assays, and the relative abundance of these bands varied in extracts from cells at different stages of EAS induction. The TAC box binding factor (TacBBF) was purified > 450-fold from crude whole-cell extracts by a combination of DNA affinity and cation exchange chromatography. The purified fractions were enriched for polypeptides of 17 and 19 kDa and the DNA binding properties of these preparations were characterized. Mutation of 2 bp in the TAC box prevented protein binding in vitro and increased both basal and elicitor-inducible gene expression 2.5-fold in transgenic tobacco plants harboring promoter-GUS fusions, consistent with the notion that this cis-element functions as a silencer or repressor of EAS gene expression.