Department of Ecology, Institute of Hydrobiology, School of Life Science and Technology, Jinan University, Guangzhou 510632, PR China.
Department of Molecular Structural Biology, Institute for Biochemistry, University of Greifswald, Felix-Hausdorff-Strasse 4, D-17489 Greifswald, Germany.
Biochem Biophys Res Commun. 2014 Jul 18;450(1):513-8. doi: 10.1016/j.bbrc.2014.06.003. Epub 2014 Jun 10.
SCO3201, a regulator of the TetR family, is a strong repressor of both morphological differentiation and antibiotic production when overexpressed in Streptomyces coelicolor. Here, we report the identification of 14 novel putative regulatory targets of this regulator using in vitro formaldehyde cross-linking. Direct binding of purified His6-SCO3201 was demonstrated for the promoter regions of 5 regulators (SCO1716, SCO1950, SCO3367, SCO4009 and SCO5046), a putative histidine phosphatase (SCO1809), an acetyltransferase (SCO0988) and the polyketide synthase RedX (SCO5878), using EMSA. Reverse transcriptional analysis demonstrated that the expression of the transcriptional regulators SCO1950, SCO4009, SCO5046, as well as of SCO0988 and RedX was down regulated, upon SCO3201 overexpression, whereas the expression of SCO1809 and SCO3367 was up regulated. A consensus binding motif was derived via alignment of the promoter regions of the genes negatively regulated. The positions of the predicted operator sites were consistent with a direct repressive effect of SCO3201 on 5 out of 7 of these promoters. Furthermore, the 2.1Å crystal structure of SCO3201 was solved, which provides a possible explanation for the high promiscuity of this regulator that might account for its dramatic effect on the differentiation process of S. coelicolor.
SCO3201 是 TetR 家族的调控因子，在变铅青链霉菌中过表达时，强烈抑制形态分化和抗生素产生。在这里，我们报告了使用体外甲醛交联鉴定该调节剂的 14 个新的潜在调控靶点。通过 EMSA 证明了纯化的 His6-SCO3201 与 5 个调节剂（SCO1716、SCO1950、SCO3367、SCO4009 和 SCO5046）、一个假定的组氨酸磷酸酶（SCO1809）、乙酰转移酶（SCO0988）和聚酮合酶 RedX（SCO5878）的启动子区域的直接结合。反转录分析表明，在 SCO3201 过表达时，转录调节因子 SCO1950、SCO4009、SCO5046 以及 SCO0988 和 RedX 的表达下调，而 SCO1809 和 SCO3367 的表达上调。通过对受负调控基因启动子区域的比对，推导出了一个保守的结合基序。预测的操纵子位点的位置与 SCO3201 对这 7 个启动子中的 5 个的直接抑制作用一致。此外，还解析了 SCO3201 的 2.1Å 晶体结构，这为该调节剂的高混杂性提供了一个可能的解释，这可能解释了它对变铅青链霉菌分化过程的显著影响。
