FUNCTIONAL-ANALYSIS OF THE TDCABC PROMOTER OF ESCHERICHIA-COLI - ROLES OF TDCA AND TDCR

The efficient expression of the tdc operon of Escherichia coli require s the products of two regulatory genes, tdcR and tdcA. We have identif ied the transcription site of tdcR by primer extension mapping and est ablished the translation start site of TdcR by mutational analysis of its reading frame. In a tdcR tdcABC deletion strain, tdcR(+) promoted high-level LacZ expression from a lambda tdcAB-lacZ lysogen and mutati ons introduced in tdcR resulted in a greater than sixfold decrease in LacZ level. In-frame deletions of tdcA also reduced LacZ expression, a nd chromosomal and plasmid-borne tdcA(+) increased the LacZ level in t dcA mutant lysogens. Interestingly, multicopy tdcA(+) plasmids introdu ced into tdcR mutant strains completely restored tdc expression. In se parate experiments we found that mutations in the tdc promoter DNA aro und positions -70, -140, and -175 greatly reduced tdc expression relat ive to that for the mild-type promoter and the tdcP mutation around -1 75 prevented multicopy tdcA(+) from rescuing tdcR mutants. Furthermore , competition experiments revealed that a wild-type promoter fragment encompassing the -175 region cloned into a plasmid reduced tdc express ion by titrating TdcA in vivo, and this effect was reversed with exces s TdcA. These results suggest that in tdcR(+) cells TdcR interacts wit h tdcP and/or TdcA to enhance tdc transcription,whereas in tdcR mutant cells a new tdcP-TdcA complex around -175 in the native promoter bypa sses the requirement for TdcR. On the basis of the accumulated data su mmarized here and elsewhere we propose that multiple transcription fac tors enhance tdc operon expression by bending and looping of the promo ter DNA to form an active transcription complex.