Ligand-induced changes in the Streptomyces lividans TipAL protein imply an alternative mechanism of transcriptional activation for MerR-like proteins.

TipAL is a Streptomyces transcriptional activator assigned to the MerR/SoxR family based both on homology within its putative DNA recognition domain and the fact that its operator binding sites lie within a region of its promoter normally occupied by RNA polymerase. The tipA gene is also independently translated as the C-terminal ligand-binding domain of TipAL (TipAS; residues 111-254). Both TipAS and TipAL share broad recognition specificity for cyclic thiopeptide antibiotics. The molecular mechanism by which TipAL catalyzes prokaryotic transcriptional activation at the tipA promoter (ptipA) in response to thiostrepton was studied using a combination of analytical ultracentrifugation (AU), circular dichroism (CD), optical waveguide lightmode spectroscopy (OWLS; a sensitive in situ binding assay), and mutational analyses. AU showed that TipAL, but not TipAS, was a dimer in solution in the presence or absence of thiostrepton. This indicated that activation of TipAL by thiostrepton was not mediated by changes in multimerization and mapped the dimerization domain to its N-terminal 110 amino acids, presumably within amino acids predicted to form a coil-coil domain (residues 77-109). CD spectra showed that TipAL had more alpha-helical content than TipAS, probably because of the presence of the additional N-terminal region. The helicity of TipAL and TipAS both increased slightly after binding thiostrepton demonstrating conformation changes upon thiostrepton binding. OWLS experiments determined the overall binding constants via measurements of association and dissociation rates for both TipA proteins and RNA polymerase with ptipA. Thiostrepton slightly enhanced the rate of specific association of TipAL with ptipA, but drastically lowered the rate of dissociation from the binding site. TipAL-thiostrepton increased the affinity of RNA polymerase for ptipA more than 10-fold. In conjunction with genetic experiments, we propose that, while there are some similarities, the mechanism by which TipAL activates transcription is distinctly different from the established MerR/SoxR paradigm.