CHARACTERIZATION OF THE TAC BOX, A CIS-ELEMENT WITHIN AN ELICITOR-INDUCIBLE SESQUITERPENE CYCLASE PROMOTER

The first unique step in the synthesis of the tobacco phytoalexin caps idiol is cyclization of farnesyl pyrophosphate catalyzed by 5-epi-aris tolochene synthase (EAS), a sesquiterpene cyclase. Earlier work demons trated that the elicitor-inducibility of this enzyme activity correspo nded to the transcriptional activation of at least one gene, EAS4, of a rather complex gene family consisting of > 10 members. To investigat e the mechanism(s) controlling expression of this gene, fragments of t he EAS4 promoter were examined for binding by proteins in nuclear and whole-cell extracts. A strong protein binding site (TAC box; ACTCTACAG TACTC) was identified between -245 and -232 by the electrophoretic mob ility shift assay, and DNAase I and methylation interference footprint ing. Several distinctly migrating bands representing protein-TAC box c omplexes were also observed in the mobility shift assays, and the rela tive abundance of these bands varied in extracts from cells at differe nt stages of EAS induction. The TAC box binding factor (TacBBF) was pu rified >450-fold from crude whole-cell extracts by a combination of DN A affinity and cation exchange chromatography. The purified fractions were enriched for polypeptides of 17 and 19 kDa and the DNA binding pr operties of these preparations were characterized. Mutation of 2 bp in the TAC box prevented protein binding in vitro and increased both bas al and elicitor-inducible gene expression 2.5-fold in transgenic tobac co plants harboring promoter-GUS fusions, consistent with the notion t hat this cis-element functions as a silencer or repressor of EAS gene expression.