Regulation of Gene Transcription

1)转录调控机制。TATA元件启动子-11位的腺嘌呤是关键的，足以发出碱基对变形的信号。大肠杆菌启动子-11位保守的A：T碱基对替换非常敏感。在体外转录与galP 1启动子有一个天然或非天然的基础上，在任何一条链的位置-11显示，只有一个嘌呤碱基没有侧基在非模板链的C2是转录有效的，无论是嘌呤与氨基在C2也不是嘧啶支持转录。在-11位无处不在的腺嘌呤中，C6位的氨基在促进转录方面不起任何作用。模板链中-11位碱基的性质（互补或非互补）也不影响转录。我们提出，腺嘌呤，成为extrahelical，相互作用的氨基酸（S）的2.3-2.4区域的σ，其中一个未取代的C2氢是关键的。2)不同超螺旋对转录的影响及其调控。DNA模板的超螺旋性对转录的影响在几种情况下有很好的记录。然而，引起任何变化所需的超螺旋的量以及施加这种影响的步骤没有系统地研究。我们研究了DNA超螺旋对转录的影响，从一组启动子上存在的质粒，通过使用一系列的拓扑异构体与不同的超螺旋密度范围从完全放松超过生理。这些拓扑异构体在基因调控蛋白的存在和不存在下的体外转录测定表明，负超螺旋对内在转录的影响因启动子而异。一些启动子，其中DNA超螺旋刺激转录，显示特定的最佳超螺旋密度，而其他人没有。结果还表明，从两个启动子的流产RNA合成量减少，全长RNA增加，增加超螺旋，首次表明全长和流产RNA合成之间的反比关系，并支持DNA超螺旋在启动子清除的作用。DNA超螺旋也可能影响RNA链的终止点。此外，改变超螺旋的量对基因调控蛋白的作用的影响表明了作用模式，这与以前的结果一致。我们的研究结果强调了DNA超螺旋在微调启动子活性中的重要性，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发生在不同的环境中。3)腺苷酸环化酶与环腺苷酸受体蛋白的相互作用。我们已经研究了腺苷酸环化酶与受体蛋白进行物理接触以有效递送环AMP用于从启动子激活转录的可能性。通过使用纯化的His标记的腺苷酸环化酶和CRP，并采用镍柱，我们已经证明了两种蛋白质之间的物理相互作用。这通过共免疫沉淀测定进一步证实。我们目前正在研究这种复合物的生物化学性质。细菌染色体结构1)A-tract簇有助于染色体中DNA的凝聚。细菌染色体包装的分子机制仍然不清楚，因为细菌缺乏核小体或其他明显的DNA压缩基本元件。在促进DNA缩合的因素中，可能是DNA分子由于其固有曲率而折叠的倾向。如前所述，基因组中的序列相关性反映了这种倾向[Trifonov和Sussman（1980）Proc. Natl Acad. Sci. USA，77，3816-3820]。

1) Mechanism of transcription regulation. Adenine at -11 position of a TATA element a promoter is critical and sufficient to signal base pair deformation. The conserved A: T base pair at the -11 position of the promoters in Escherichia coli is very sensitive to substitutions. In vitro transcription with the galP1 promoter having a natural or unnatural base in either strand at position -11 showed that only a purine base with no side group at C2 in the nontemplate strand is transcriptionally potent; neither a purine with amino group at C2 nor a pyrimidine support transcription. The amino group at C6 in the omnipresent adenine at -11 does not play any role in promoting transcription. The nature of the base, complementary or noncomplementary, at -11 in the template strand also does not influence transcription. We proposed that the adenine, by becoming extrahelical, interacts with an amino acid(s) of the 2.3-2.4 region of sigma for which an unsubstituted C2 hydrogen is critical. 2) Effect of varying supercoiling on transcription and its regulation. The effect of superhelicity of DNA templates on transcription is well documented in several cases. However, the amount of supercoiling that is needed to bring about any changes and the steps at which such effects are exerted were not systemically studied. We investigated the effect of DNA supercoiling on transcription from a set of promoters present on a plasmid by using a series of topoisomers with different superhelical densities ranging from totally relaxed to more than physiological. In vitro transcription assays with these topoisomers in the absence and presence of gene regulatory proteins showed that the effect of negative supercoiling on intrinsic transcription varies from promoter to promoter. Some of the promoters, in which DNA superhelicity stimulated transcription, displayed specific optima of superhelical density while others did not. The results also showed that the amount of abortive RNA synthesis from two of the promoters decreased and full-length RNA increased with increasing supercoiling, indicating for the first time an inverse relationship between full-length and abortive RNA synthesis and supporting a role of DNA superhelicity in promoter clearance. DNA supercoiling might also influence the point of RNA chain termination. Furthermore, the effect of varying the amount of supercoiling on the action of gene regulatory proteins suggested the mode of action, which is consistent with previous results. Our results underscore the importance of DNA supercoiling in fine-tuning promoter activities, which should be relevant in cell physiology given that local changes in chromosomal supercoiling must occur in different environments. 3) Interaction of adenylate cyclase and cyclic AMP receptor protein. We have investigated the possibility that adenylate cyclase makes physical contact with the receptor protein for efficient delivery of cyclic AMP for activation of transcription from a promoter. By using purified His-tagged adenylate cyclase and CRP and employing Nickel column, we have demonstrated a physical interaction between the two proteins. This was further confirmed by co-immuno precipitation assays. We are currently investigating the biochemical properties of this complex. Bacterial Chromosome Structure. 1) A-tract cluster facilitates DNA condensation in chromosome. Molecular mechanisms of bacterial chromosome packaging are still unclear, as bacteria lack nucleosomes or other apparent basic elements of DNA compaction. Among the factors facilitating DNA condensation may be propensity of the DNA molecule for folding due to its intrinsic curvature. As suggested previously, the sequence correlations in genome reflect such a propensity [Trifonov and Sussman (1980) Proc. Natl Acad. Sci. USA, 77, 3816-3820].