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大多数在正调控中功能有缺陷的突变 OccR 蛋白使操纵子 DNA 保持在锁定的高角度弯曲状态。

Most mutant OccR proteins that are defective in positive control hold operator DNA in a locked high-angle bend.

机构信息

Department of Microbiology, 360A Wing Hall, Cornell University, Ithaca, NY 14853, USA.

出版信息

J Bacteriol. 2011 Oct;193(19):5442-9. doi: 10.1128/JB.05352-11. Epub 2011 Jul 29.

Abstract

OccR is a LysR-type transcriptional regulator of Agrobacterium tumefaciens that positively regulates the octopine catabolism operon of the Ti plasmid. Positive control of the occ genes occurs in response to octopine, a nutrient released from crown gall tumors. OccR also functions as an autorepressor in the presence or absence of octopine. OccR binds to a site between occQ and occR in the presence or absence of octopine, although octopine triggers a conformational change that shortens the DNA footprint and relaxes a DNA bend. In order to determine the roles of this conformational change in transcriptional activation, we isolated 11 OccR mutants that were defective in activation of the occQ promoter but were still capable of autorepression. The mutations in these mutants spanned most of the length of the protein. Two additional positive-control mutants were isolated using site-directed mutagenesis. Twelve mutant proteins displayed a high-angle DNA bend in the presence or absence of octopine. One mutant, the L26A mutant, showed ligand-responsive DNA binding similar to that of wild-type OccR and therefore must be impaired in a subsequent step in activation.

摘要

OccR 是一种 LysR 型转录调节因子,能够正向调控农杆菌 Ti 质粒上的 octopine 分解代谢操纵子。OccR 在响应 octopine 时,对 occ 基因进行正调控,而 octopine 是冠瘿瘤释放的一种营养物质。OccR 无论有无 octopine 的存在,都可以作为自身的阻遏物。OccR 在有或没有 octopine 的情况下,都能结合 occQ 和 occR 之间的一个位点,尽管 octopine 会引发构象变化,从而缩短 DNA 足迹并放松 DNA 弯曲。为了确定这种构象变化在转录激活中的作用,我们分离了 11 个 OccR 突变体,这些突变体在激活 occQ 启动子方面存在缺陷,但仍能进行自身抑制。这些突变体中的突变横跨蛋白的大部分长度。通过定点诱变,我们还分离了另外两个正调控突变体。12 种突变蛋白在有或没有 octopine 的情况下都表现出高角度 DNA 弯曲。一个突变体,即 L26A 突变体,显示出与野生型 OccR 相似的配体响应 DNA 结合,因此必须在激活的后续步骤中受到损伤。

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