Characterization of LeuO and additional LysR-type transcription regulators in Escherichia coli

大肠杆菌中 LeuO 和其他 LysR 型转录调节因子的表征

• 批准号: 392159619
• 负责人: Professorin Dr. Karin Schnetz
• 金额: --
• 依托单位: Arbeitsgruppe Bacterial Genetics
• 依托单位国家: 德国
• 项目类别: Research Grants
• 财政年份: 2017
• 资助国家: 德国
• 起止时间: 2016-12-31 至 2022-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/392159619?language=en
• 关键词: Characterization; LeuO; additional; LysR; type

LysR-type transcription regulators (LTTRs) constitute the most abundant family of transcription regulators in Bacteria and are implicated in regulation of a broad spectrum of processes related to stress responses, metabolism, and pathogenicity. LTTRs consist of an N-terminal winged helix-turn-helix (wHTH) DNA-binding domain that is connected with a helical linker to a C-terminal effector-binding domain (EBD). LTTRs are tetramers which contain two dimeric wHTH-linker domains and specifically contact DNA at two sites. Effector-binding induces a structural change that triggers a shift of one of these contacts along the DNA (sliding dimer mechanism) and thus a concomitant change in the binding pattern which is relevant for the effector-specific regulation of target genes, when the LTTR is present at a physiological low level. However, the sliding dimer mechanism of regulation and the dependence of the specificity on the cellular level has not been considered in many studies on LTTRs of pleiotropic function. With this proposal the pleiotropic LTTR LeuO in Escherichia coli, as well as additional LTTRs, shall be functionally characterized in respect to activity, mechanism of transcription regulation, and pleiotropic function. LeuO regulates more than 100 target loci and is considered an antagonist of the global nucleoid-associated repressor proteins H-NS and StpA. However, up to date it is unknown how LeuO activity is modulated, how LeuO specifically binds DNA, and how LeuO regulates transcription and acts as H-NS and StpA antagonist. In preliminary work we have solved the crystal structure of the LeuO-EBD and isolated hyperactive mutants that presumably mimic an effector-induced state. With this proposal we plan to characterize the DNA-binding specificity by comparing wild-type LeuO, hyperactive LeuO mutants, and the dimeric wHTH-linker domain, which is sufficient for activation of target loci. In addition, ChIP-seq and RNA-seq with a hyperactive LeuO variant will be used to re-evaluate the spectrum of target loci. Second, we wish to establish a structure-function relationship of LeuO by solving the crystal structure of full length LeuO and by a mutational analysis which will include genetic and biochemical assays. Replacement of specific amino acid residues by crosslinkable unnatural amino acids shall identify interactions relevant for transcriptional regulation (as with RNA-polymerase and/or H-NS and StpA). Third, the mechanism of regulation as H-NS and StpA antagonist shall be addresses in vivo. To this end, LeuO will be tagged with repeats of a split-superfolder-gfp epitope (for fluorescence signal enhancement) and then used for co-localization studies with H-NS. With these two approaches (crosslinking and co-localization) the question whether LeuO co-binds with H-NS or displaces it from the DNA may be clarified. This working pipeline, established for LeuO, will be applied for characterization of the functional role of other LTTRs in E. coli.

LysR-type转录调节因子(Lttrs)是细菌中含量最丰富的转录调节因子家族，参与调节一系列与应激反应、代谢和致病相关的过程。Lttrs由一个N-末端有翼螺旋-转角螺旋(WHTH)DNA结合域组成，它与C-末端效应器结合域(EBD)的螺旋连接物相连。Lttrs是四聚体，它包含两个二聚体的wHTH-连接结构域，并在两个位点特异性地接触DNA。当LTTR处于生理低水平时，效应器结合会引起结构变化，触发其中一个接触沿DNA(滑动二聚体机制)移动，从而导致与靶基因的效应器特异性调节相关的结合模式的改变。然而，在许多关于多效性功能的研究中，还没有考虑到滑动二聚体的调节机制和特异性对细胞水平的依赖性。根据这一建议，将从活性、转录调控机制和多效性功能方面对大肠杆菌中的多效性LTTRLeuO以及额外的LTTrs进行功能表征。LeuO调节100多个靶点，被认为是全球核类相关抑制蛋白H-NS和StpA的拮抗剂。然而，到目前为止，还不清楚LeuO的活性是如何调节的，LeuO是如何与DNA特异结合的，以及LeuO是如何调节转录和作为H-NS和StpA拮抗剂的。在初步工作中，我们已经解决了LeuO-EBD的晶体结构，并分离出了可能模拟效应器诱导状态的超活性突变体。根据这一建议，我们计划通过比较野生型LeuO、超活性LeuO突变体和足以激活靶基因的二聚体wHTH-连接结构域来表征DNA结合的特异性。此外，还将使用具有超活性LeuO变体的CHIP-SEQ和RNA-SEQ来重新评估目标基因座的谱。其次，我们希望通过解决全长LeuO的晶体结构和包括遗传和生化分析在内的突变分析来建立LeuO的结构-功能关系。用可交联的非天然氨基酸取代特定的氨基酸残基，应确定与转录调控有关的相互作用(如与RNA聚合酶和/或H-NS和StpA)。第三，作为H-NS和StpA拮抗剂的调节机制应在体内解决。为此，LeuO将被标记为重复的分裂超文件夹-GFP表位(用于荧光信号增强)，然后用于与H-NS的共同定位研究。有了这两种方法(交联法和共定位法)，LeuO是与H-NS共结合还是将其从DNA上置换的问题可能会得到澄清。这条为LeuO建立的工作管道将用于鉴定其他Lttrs在大肠杆菌中的功能作用。