Regulation of Gene Transcription

我们以大肠杆菌中的半乳糖操纵子为实验系统，研究了转录受激活子、抑制子、终止子和反终止子调控的机制。Gal操纵子由两个串联启动子P1和P2转录，这两个启动子受几种调控蛋白以不同方式调控。我们报告了今年的几项发现。 关于DNA环化要求的研究 我们之前已经证明，HU以比其特征良好的非特异性DNA结合更高的亲和力与GAL DNA环的顶端结合。这种高亲和力结合完全依赖于GalR与两个操作符的结合(协作性)。现在我们证明了协作性是特定的GalR-Hu相互作用的结果。这种相互作用通过免疫共沉淀以及相互作用缺陷HU突变体的分离和鉴定得到证实。我们得出结论，GalR将HU“携带”到HU结合部位进行DNA环化。 我们用原子力显微镜研究了GalR/Hu介导的DNA环，证实了环的发生是由DNA中两个GalR结合部位的“反平行”而不是“平行”的几何构型引起的。 我们通过遗传分析确定了DNA环化过程中GalR二聚体-二聚体相互作用的界面。通过分离和鉴定GalR与DNA结合但不四聚体的突变体，鉴定了一组位于新月形区域内二聚体表面的五个氨基酸残基。我们先前证明，GalR-GalR相互作用创建DNA环是通过结合DNA弯曲蛋白Hu来实现的，该蛋白大约在环的顶端。HU在DNA环中的作用仅表现为稳定GalR四聚化。我们分离并鉴定了GalR突变体，它们形成了更强的四聚体，不需要HU来环化和抑制，证实了HU的作用只是辅助，而不是必需的。 -11碱基对在启动子“融化”中的作用 我们发现，在DNA链分离中，位于启动子非模板链-11位的无处不在的腺嘌呤是DNA链分离的主要碱基。这个位置的A：T碱基对首先扭曲。这一点的突变改变阻止了启动子中其他地方的链分离。我们进一步证明了嘌呤碱基上未被取代的C2氢在-11主位置对碱基对变形的信号是关键的和充分的。 激活蛋白抑制启动子清除的转录抑制作用 C反应蛋白：C反应蛋白通过与转录起始点上游-61个碱基结合来刺激乳胶启动子的转录。如上所述，我们分离到一个不依赖于CRP的lac启动子变异体，该变异体具有很强的活性。我们通过在-10区上游设置一个5‘TG3’序列，将这个不需要CRP转录的强大lac启动子转化为扩展的-10启动子类型。我们发现，CRP与-61位的结合抑制了Extended-10启动子的转录。对转录产物的分析表明，CRP阻止了启动子清除的步骤。在CRP存在的情况下，产生性RNA合成减少，流产的RNA合成增加。这一发现与我们之前提出的调控因子通过与RNA聚合酶的差异接触发挥作用的模型是一致的。在这种情况下，CRP与RNA聚合酶的接触降低了激活能，使其达到从“空转”复合体到拉长复合体的过渡状态。 DNA超螺旋对转录启动的影响 与其他DNA交易一样，许多基因的转录也受到DNA超螺旋的影响。然而，带来任何变化所需的超级线圈的数量以及产生这种影响的步骤并没有得到系统的研究。我们研究了DNA超螺旋对一组启动子转录的影响，这些启动子包括GAL的P1和P2启动子，存在于一个质粒上，使用了一系列具有不同超螺旋密度的拓扑异构体，从完全松弛到超过生理密度。在没有和存在基因调控蛋白的情况下，对这些拓扑异构体进行的体外转录实验研究了全长和流产(如果适用)RNA合成，结果表明，负超螺旋对内在转录的影响因启动子而异。在DNA超螺旋刺激转录的启动子(包括P1和P2)中，有些启动子显示出超螺旋密度的特异最佳值，而另一些则没有。结果还表明，两个GAL启动子产生的流产转录物的数量随着负超螺旋的增加而减少，这首次表明全长RNA合成与流产RNA合成之间存在相反的关系，从而支持了DNA超螺旋在启动子清除中的作用。 不同数量的超级卷曲对基因调控蛋白CRP和GalR的作用表明了各自调控蛋白的作用模式(包括激活和抑制)，这与以前的作用模型一致。我们的结果强调了DNA超螺旋在微调启动子活性方面的重要性，这在细胞生理学中应该是相关的，因为染色体超螺旋的局部变化必须在不同的环境中发生。

We study the mechanism by which transcription is regulated by activators, repressors, terminators, and anti-terminators by using the galactose opern in Escherichia coli as an experimental system. The gal operon is transcribed by two tandem promoters, P1 and P2, which are regulated in different ways by several regulatory proteins. We report several discoveries made this year. Studies on Requirements of DNA Looping We previously showed that HU binds to the apex of the gal DNA loop at affinities much higher than its well characterized nonspecific DNA binding. This high affinity binding was totally dependent upon the binding of GalR to the two operators (cooperativity). Now we showed that the cooperativity is the result of a specific GalR-HU interaction. The interaction was demonstrated by co-immunoprecipitation, as well as by isolation and characterization of interaction defective HU mutants. We concluded that GalR "piggybacks" HU to the HU binding site for DNA looping. We studied the GalR/HU mediated DNA looping by atomic force microscopy and confirmed that looping occurs by an "antiparallel" not a "parallel" geometry of the two GalR binding sites in DNA. We identified the GalR dimer-dimer interaction interface during DNA looping by genetic analysis. A set of five amino acid residues, located in a dimer surface within a "crescent" area were identified by isolating and characterizing mutants of GalR that bind to DNA but do not tetramerize. We previously showed that the GalR-GalR interaction to create a DNA loop is facilitated by binding of a DNA bending protein, HU, approximately in the apex of the loop. The role of HU in DNA looping was shown only to stabilize the GalR tetramerization. We isolated and characterized GalR mutants, which form stronger tetramers and which do not need HU to loop and repress, confirming that the role of HU is only to aid and is not essential. Role of -11 Base Pair in Promoter"Melting" We identified the omnipresent adenine at the -11 position of the non-template strand of a promoter as the "master" base in DNA strand separation. The A:T base pair at this position distorts first. Mutational changes at this site prevent strand separation everywhere else in the promoter. We further showed an unsubstituted C2 hydrogen of a purine base is critical and sufficient at the -11 master position to signal base pair deformation. Transcription repression by inhibition of promoter clearance by the activator protein CRP: CRP stimulates transcription from the lac promoter by binding to a site that is -61 bp upstream of the transcription start point. As mentioned above, we isolated a CRP independent lac promoter variant that showed a strong activity. We converted this strong lac promoter, which does not need CRP for transcription, into an extended -10 promoter type by setting a 5'TG3' sequence one base pair upstream of the -10 region. We discovered that CRP binding to the -61 site repressed transcription from the extended -10 promoter. Analysis of transcription products demonstrated that CRP blocks the step of promoter clearance. In the presence of CRP, productive RNA synthesis decreased and abortive RNA synthesis increased. This finding is consistent with our previously proposed model that a regulator acts by differential contacts with RNA polymerase. In this case, CRP contact with RNA polymerase lowers the activation energy to reach the transition state of the "idling" complex to the elongating complex. Effect of DNA supercoiling on transcription initiation Transcription of many genes, like other DNA transactions, is affected by DNA supercoiling. However, the amount of supercoiling that is needed to bring about any changes and the steps at which such affects are exerted were not systematically studied. We investigated the effect of DNA supercoiling on transcription from a set of promoters, including the P1 and P2 promoters of gal, present on a plasmid by using a series of its topoisomers with different superhelical densities ranging from totally relaxed to more than physiological. In vitro transcription assays performed on these topoisomers to study full-length and abortive (when applicable) RNA synthesis in the absence and presence of gene regulatory proteins showed that the effect of negative supercoiling on intrinsic transcription varies from promoter-to-promoter. Among those promoters, which included P1 and P2, in which DNA superhelicity stimulated transcription, some displayed specific optima of superhelical density while others did not. The results also showed that the amounts of abortive transcripts made from the two gal promoters decreased with increased negative supercoiling, suggesting for the first time an inverse relationship between full-length and abortive RNA synthesis, thus, supporting a role of DNA superhelicity in promoter clearance. The effect of varying amount of supercoiling on the action of gene regulatory proteins, CRP and GalR, suggested mode of action (both activation and repression) of the respective regulators, which are consistent with previous models of their action. Our results underscored the importance of DNA supercoiling in fine tuning of promoter activities which should be relevant in cell physiology given that local changes in chromosomal supercoiling must occur in different environments.