Ligand-Induced Changes in the Streptomyces lividans TipAL Protein Imply an Alternative Mechanism of Transcriptional Activation for MerR-Like Proteins

Abstract

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.

Structural basis and dynamics of multidrug recognition in a minimal bacterial multidrug resistance system

“…Antibiotic binding to TipAL increases the affinity to its operator site and stabilizes binding of the RNA polymerase to the ptipA promoter (27). TipAL binds DNA as a dimer and presumably, by analogy to MerR (28,29,34), might activate transcription of tipA by twisting the DNA.…”

“…As the N-terminal part of TipAS connects to the DNA binding domain in the transcriptional regulator TipAL, its folding upon binding very likely also constitutes the mechanical signal by which antibiotic binding induces expression of tipA and possibly of other MDR genes (24,25). The conformational selection of the unfolded N terminus to a well-defined, almost identical structure in all three TipAS complexes would then position the DNA binding domain in an appropriate way to bind promoter DNA with higher affinity and recruit RNA polymerase to the complex (27). This specific positioning of the DNA binding domain and the subsequent induction of transcription needs to emerge from the specific thiopeptide recognition motif.…”

“…The thiopeptide antibiotics induce expression of the tipA gene as two alternate in-frame translation products: a long protein, TipAL (253 aa), and a short protein, TipAS (144 aa), which also constitutes the C-terminal part of TipAL (24,26). TipAL belongs to the MerR family of stress response regulators, characterized by their homologous N-terminal DNA-recognition domains; however, its mechanism of promoter activation has distinctive characteristics (27). Their highly diverse C-terminal domains (19,20,28) recognize a wide variety of ligands ranging from divalent metal ions to large antibiotics (29).…”

Functional Analysis of the Carboxy-Terminal Region of Bacillus subtilis TnrA, a MerR Family Protein

“…The N-and C-terminal domains are linked together by a long ␣-helix that forms an antiparallel coiled-coil structure that is involved in dimerization ( Fig. 1) (6,7,9,19,21,53).…”

Isolation and Characterization of a Rolling-Circle-Type Plasmid from Rhodococcus erythropolis and Application of the Plasmid to Multiple-Recombinant-Protein Expression

“…The pTip vectors are tightly regulated expression vectors containing a tipA promoter (P tipA ) (4,14), from which protein expression is induced by the antibiotic reagent thiostrepton (26). In our previous report, we showed that pTip vectors mediate heterologous and homologous protein expression, as they contain multiple cloning sites for 11 restriction enzyme sites and a hexahistidine (six-His) tag sequence and they work over a wide temperature range, from 4 to 35°C.…”