Ligand-induced changes in the Streptomyces lividans TipAL protein imply analternative 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 a nd the fact that its operator binding sites lie within a region of its prom oter normally occupied by RNA polymerase. The tipA gene is also independent ly translated as the C-terminal ligand-binding domain of TipAL (TipAS; resi dues 111-254). Both TipAS and TipAL share broad recognition specificity for cyclic thiopeptide antibiotics. The molecular mechanism by which TipAL cat alyzes prokaryotic transcriptional activation at the tipA promoter (ptipA) in response to thiostrepton was studied using a combination of analytical u ltracentrifugation (AU), circular dichroism (CD), optical waveguide lightmo de, spectroscopy (OWLS; a sensitive in situ binding assay), and mutational analyses. AU showed that TipAL, but not TipAS, was a dimer in solution in t he presence or absence of thiostrepton. This indicated that activation of T ipAL by thiostrepton was not mediated by changes in multimerization and map ped the dimerization domain to its N-terminal 110 amino acids, presumably w ithin amino acids predicted to form a coil-coil domain (residues 77-109). C D spectra showed that TipAL had more alpha -helical content than TipAS, pro bably because of the presence of the additional N-terminal region. The heli city of TipAL and TipAS both increased slightly after binding thiostrepton demonstrating conformation changes upon thiostrepton binding. OWLS experime nts determined the overall binding constants via measurements of associatio n and dissociation rates for both TipA proteins and RNA polymerase with pti pA. Thiostrepton slightly enhanced the rate of specific association of TipA L with ptipA, but drastically lowered the rate of dissociation from the bin ding site. TipAL-thiostrepton increased the affinity of RNA polymerase for ptipA more than 10-fold. In conjunction with genetic experiments, we propos e that, while them are some similarities, the mechanism by which TipAL acti vates transcription is distinctly different from the established MerR/SoxR paradigm.