Regulation of Gene Transcription

1)转录调控机制。TATA元件启动子-11位置的腺嘌呤对碱基对变形至关重要，足以发出信号。大肠杆菌启动子-11位上保守的A: T碱基对对取代非常敏感。在体外转录中，galP1启动子在-11位的任一链上都有天然或非天然碱基，结果表明，只有非模板链上C2处没有侧基的嘌呤碱基具有转录效力；既不是具有C2氨基的嘌呤也不是支持转录的嘧啶。无所不在的腺嘌呤-11位点C6的氨基对促进转录没有任何作用。碱基的性质，互补或非互补，在模板链的-11也不影响转录。我们提出，腺嘌呤通过变成外螺旋，与sigma 2.3-2.4区域的氨基酸相互作用，其中未取代的C2氢是至关重要的。2)不同超卷曲度对转录及其调控的影响。DNA模板的超螺旋对转录的影响在几个案例中得到了很好的证明。然而，产生任何变化所需的超卷曲量以及施加这种影响的步骤并没有得到系统的研究。我们利用一系列不同超螺旋密度的拓扑异构体研究了DNA超螺旋对质粒上一组启动子转录的影响，这些超螺旋密度从完全放松到超过生理。这些拓扑异构体在缺乏和存在基因调控蛋白的情况下的体外转录分析表明，负超旋对内在转录的影响因启动子而异。在一些启动子中，DNA超螺旋刺激转录，显示出特定的超螺旋密度，而其他启动子则没有。结果还表明，随着超螺旋度的增加，两个启动子的流产RNA合成量减少，全长RNA合成量增加，这首次表明全长RNA合成与流产RNA合成呈反比关系，并支持DNA超螺旋度在启动子清除中的作用。DNA的超卷曲也可能影响RNA链的终止点。此外，改变超卷曲量对基因调控蛋白作用的影响提示了作用模式，这与先前的结果一致。我们的研究结果强调了DNA超卷绕在微调启动子活动中的重要性，这应该与细胞生理学有关，因为染色体超卷绕的局部变化必须在不同的环境中发生。腺苷酸环化酶与环AMP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的可能性，以有效地递送环AMP，从而激活启动子的转录。通过纯化的his标记的腺苷酸环化酶和CRP，并采用镍柱，我们证明了两者之间的物理相互作用。共免疫沉淀试验进一步证实了这一点。我们目前正在研究这种复合物的生化特性。细菌染色体结构：1)a束簇有利于染色体内DNA的凝聚。细菌染色体包装的分子机制尚不清楚，因为细菌缺乏核小体或其他明显的DNA压缩的基本元素。在促进DNA缩聚的因素中，DNA分子由于其固有的曲率而具有折叠的倾向。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.][j].中国生物医学工程学报，2016,33(2):387 - 398。

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].