Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:2.7.7.6 (RNA polymerase)
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Thiostrepton induced the expression of four proteins (17, 19, 30, and 56 kilodaltons) of unknown function in Streptomyces lividans. The chromosomal gene which encoded the 19-kilodalton protein (tipA) was cloned and sequenced. Transcription of the tipA promoter was induced at least 200-fold by thiostrepton. The tipA 200-fold by thiostrepton. The tipA transcriptional start site (located by S1 mapping and primer extension experiments) was preceded by a 45-base-pair imperfect inverted-repeat sequence which included the -10 and -35 regions of the promoter. Under noninducing conditions in vivo, this might form a cruciform structure which is not recognized by RNA polymerase. A 143-base-pair fragment including this region was cloned into a promoter probe vector, pIJ486. In this plasmid, pAK114, the thiostrepton-inducible tipA promoter controlled the expression of a kanamycin resistance gene encoding an aminoglycoside phosphotransferase. As little as 1 ng of thiostrepton spotted on a lawn of S. lividans(pAK114) induced kanamycin-resistant growth. Other thiostreptonlike antibiotics also induced tipA, but structurally unrelated antibiotics which inhibit translation had no effect. In S. lividans, the promoter could be induced by thiostrepton during either growth or stationary phase. The tipA promoter should be a valuable tool for expression studies in streptomycetes.
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PMID:Thiostrepton-induced gene expression in Streptomyces lividans. 253 19

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.
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PMID:Ligand-induced changes in the Streptomyces lividans TipAL protein imply an alternative mechanism of transcriptional activation for MerR-like proteins. 1166 32