DNA-BINDING EXERTED BY A BACTERIAL GENE REGULATOR WITH AN EXTENSIVE COILED-COIL DOMAIN

Although quite common in the eukaryotic cell, bacterial proteins with an extensive coiled-coil domain are still relatively rare. One of the few thus far documented examples, TlpA from Salmonella typhimurium, is characterized by a remarkably long (250 amino acids) alpha-helical co iled-coil domain. Herein, we demonstrate that TlpA is a novel, sequenc e-specific DNA-binding protein. Several tlpA deletion mutants have bee n constructed, and their corresponding protein products were purified and tested for DNA binding. Two of the mutant proteins were shown to b e deficient in DNA binding. Both mutants were analyzed by circular dic hroism and electron microscopy, supporting the notion that mutant prot eins were largely intact despite lacking the amino acid residues neces sary for DNA binding. In vivo studies with transcriptional tlpA-lacZ f usions demonstrated that TlpA acts as a repressor. Using the repressor phenotype as a readout, the chain exchange previously described in vi tro could also be confirmed in vivo. We believe the coiled-coil domain acts not only as a dimerization interface but could also serve a role as a flexible modulator of the protein-DNA interaction.