Keshav Aditi, Murarka Pooja, Srivastava Preeti
Department of Biochemical Engineering & Biotechnology, Indian Institute of Technology Delhi, New Delhi, India.
Department of Biochemical Engineering & Biotechnology, Indian Institute of Technology Delhi, New Delhi, India.
Gene. 2022 Feb 5;810:146061. doi: 10.1016/j.gene.2021.146061. Epub 2021 Nov 11.
The dsz operon responsible for the biodesulfurization of organosulfurs is under the control of a 385 bp long promoter. Recently, a TetR family protein was identified which served as an activator of operon. Here we report that the TetR family protein (WP_058249973.1), named DszGR can specifically activate the dsz operon. Direct binding of the DszGR to DNA was observed at single molecule level by AFM. It was found that the binding of DszGR to the promoter DNA induces a bend by about ∼40-50° degrees which may not be enough for the activation of the promoter. Thus, bendability in the promoter sequence was analyzed. The results show that the promoter has a curvature at around -235 and -200 bp with respect to dszA start codon. On mutating this region, a decrease in activity of the promoter was observed. Our results suggest that the DszGR protein binds to the upstream sequences and induces a bend, which is facilitated by further bending of the DNA which is required for dsz promoter activity. IHF binding site present in the promoter, and a significant reduction in desulphurization activity in the absence of either IHF subunits, suggested role of IHF in regulation of the dsz operon.
负责有机硫生物脱硫的dsz操纵子受一个385 bp长的启动子控制。最近，鉴定出一种TetR家族蛋白，它作为操纵子的激活剂。在此我们报告，名为DszGR的TetR家族蛋白（WP_058249973.1）可特异性激活dsz操纵子。通过原子力显微镜在单分子水平观察到DszGR与DNA的直接结合。发现DszGR与启动子DNA的结合诱导约40 - 50°的弯曲，这可能不足以激活启动子。因此，对启动子序列的弯曲性进行了分析。结果表明，相对于dszA起始密码子，启动子在约-235和-（此处原文可能有误，推测为-200）bp处有一个曲率。对该区域进行突变时，观察到启动子活性降低。我们的结果表明，DszGR蛋白与上游序列结合并诱导弯曲，而DNA的进一步弯曲促进了这种结合，这是dsz启动子活性所必需的。启动子中存在整合宿主因子（IHF）结合位点，并且在缺乏任何一个IHF亚基时脱硫活性显著降低，这表明IHF在dsz操纵子的调控中发挥作用。
