Mechanisms of LysR-type Protein-Mediated Regulation of Gene Expression

9723452 Hatfield目前，人们的注意力主要集中在理解DNA模板的超螺旋密度如何影响启动子的转录效率。 在细菌中，已经表明转录过程改变染色体的局部超螺旋密度;在转录RNA聚合酶之前产生正DNA超螺旋，在其之后产生负DNA超螺旋。 此外，转录RNA聚合酶后产生的局部负DNA超螺旋可以激活附近趋异启动子位点的转录起始。 尽管趋异启动子排列在所有生物体中是常见的，但这些趋异启动子的生理学意义尚未完全理解。 在细菌中，基因表达的最常见的正调节因子是蛋白质的LysR家族。 LysR家族的成员被表征为序列相关的、共诱导物响应的转录激活蛋白，其激活靶基因的转录并自动调节其自身的合成。 IlvY蛋白是LysR家族蛋白，其负调节其自身结构基因ilvY的表达，并且在小分子共诱导剂存在下，正调节编码平行L-异亮氨酸、L-缬氨酸生物合成途径的第二种酶的发散转录的ilvC基因的表达。 最近已经证明，这种转录的激活受到DNA模板的超螺旋密度的显著影响。 基于这一观察结果和其他初步证据，已经假设随着共诱导物水平的增加，更多的共诱导物结合到已形成的ilvY-操纵基因DNA复合物并进一步激活ilvC转录。 这种增加的ilvC启动子活性在其尾流中产生负超螺旋，并增强附近的趋异ilvPY启动子的转录活性。 这种增加的ilvPY启动子活性导致更高的IlvY产量和在串联操纵基因位点更大的IlvY占据。 在此条件下，由于在串联操纵基因位点处较高的IlvY占据率，ilvC基因可被诱导至较高的最大表达水平。 因此，趋异ilvPC和ilvPY启动子之间的这种转录偶联提供了灵敏且能量经济的机制，以用底物诱导物水平调节ilvY和异构还原酶（ilvC基因产物）的产生，所述底物诱导物水平反映了细胞对支链氨基酸生物合成的异构还原酶活性的需求。 这项研究将测试这种转录偶联假说的预测，并阐明蛋白质-蛋白质和蛋白质DNA相互作用的重要性，从ilvC基因的转录的ilvY蛋白介导的激活。 预计这项研究的结果将为理解DNA超卷曲依赖性结构在基因表达中的作用提供基础信息，将提高目前对其他LysR型基因激活剂作用机制的理解水平，并且将是低等和高等真核系统中不同启动子系统研究的普遍兴趣。 目前，人们的注意力都集中在理解DNA超卷曲依赖（染色质）结构对基因表达的作用上。 在细菌中，基因表达的最常见的正调节因子是蛋白质的LysR家族，其激活靶基因的转录并自动调节其自身的合成。 本研究将为理解DNA超卷曲依赖结构在基因表达中的作用提供基础信息，将提高目前对其他LysR型基因激活剂作用机制的理解水平，并将对低等和高等真核系统中不同启动子系统的研究产生普遍兴趣。 ***

9723452 Hatfield Much attention is currently being focused toward understanding how the transcriptional efficiencies of promoters are influenced by the superhelical density of the DNA template. In bacteria it has been shown that the transcription process alters the local super helical density of the chromosome; positive DNA supercoils are generated in front of a transcribing RNA polymerase and negative DNA supercoils are generated in its wake. Moreover, the local negative DNA supercoiling generated behind a transcribing RNA polymerase can activate transcription initiation from a nearby divergent promoter site. Although divergent promoter arrangements are common in all organisms, the physiological significance of these divergent promoters is not fully understood. In bacteria, the most common positive regulators of gene expression are the LysR family of proteins. Members of the LysR family are characterized as sequence-related, co-inducer responsive, transcriptional activator proteins that activate transcription of a target gene(s) and autoregulate their own synthesis. The IlvY protein is a LysR family protein that negatively regulates the expression of its own structural gene, ilvY, and, in the presence of a small molecule co-inducer, positively regulates the expression of a divergently transcribed ilvC gene which encodes the second enzyme of the parallel L-isoleucine, L-valine biosynthetic pathway. It recently has been demonstrated that this activation of transcription is significantly influenced by the superhelical density of the DNA template. On the basis of this observation and other preliminary evidence, it has been hypothesized that as co-inducer levels increase more co-inducer binds to a performed IlvY-operator DNA complex and further activates ilvC transcription. This increased ilvC promoter activity generates negative supercoils in its wake and enhances the transcriptional activity of the nearby, divergent, ilvPY promoter. This increased ilvPY promoter activity results in hig her IlvY production and greater IlvY occupancy at the tandem operator sites. Under this condition the ilvC gene can be induced to a higher maximal level of expression because of higher IlvY occupancy at the tandem operator sites. Thus, this transcriptional-coupling between the divergent ilvPC and ilvPY promoters provides a sensitive and energetically economical mechanism to adjust IlvY and isomeroreductase (ilvC gene product) production with substrate-inducer levels which reflect the demands of the cell for isomeroreductase activity for branched chain amino acid biosynthesis. This research will test the predictions of this transcriptional-coupling hypothesis and to elucidate the protein-protein and protein DNA interactions important for IlvY protein-mediated activation of transcription from the ilvC gene. It is anticipated that the results of this research will provide fundamental information for understanding the roles of DNA supercoiling-dependent structures in gene expression, will enhance the current level of understanding of the mechanism of action for other LysR-type gene activators, and will be of general interest for investigations of divergent promoter systems in lower and higher eukaryotic systems. %%% Much attention is currently being focused toward an understanding of the roles of DNA supercoiling-dependent (chromatin) structures on gene expression. In bacteria, the most common positive regulators of gene expression are the LysR family of proteins which activate transcription of a target gene(s) and autoregulate their own synthesis. This research will provide fundamental information for understanding the roles of DNA supercoiling-dependent structures in gene expression, will enhance the current level of understanding of the mechanism of action for other LysR-type gene activators, and will be of general interest for investigations of divergent promoter systems in lower and higher eukaryotic system. ***