DNA Deformations and Interactions with Proteins

DNA 变形和与蛋白质的相互作用

• 批准号: 6762004
• 负责人: Victor Zhurkin
• 金额: --
• 依托单位: DIVISION OF BASIC SCIENCES - NCI
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6762004
• 关键词: DNA; DNA binding protein; X ray crystallography; chemical structure function; computer simulation; conformation; intermolecular interaction; nucleic acid sequence; nucleic acid structure; p53 gene /protein; protein folding; protein sequence; pyrimidine dimers; structural biology; transcription factor

Completion of the human DNA sequencing brings new emphasis on the structural aspects of the genome functioning. In the post-genomic era, it becomes more important to understand the molecular mechanisms of gene regulation (usually achieved through the protein-nucleic acid interactions). In most cases, the structural analysis of the regulatory nucleoprotein complexes requires usage of unconventional approaches, as they are too large for NMR and X-ray crystallography. The project presented below is aimed at application of the new methods, developed by us recently, to three specific problems: assembly of prokaryotic repressosomes; stabilization and regulation of the transription elongation complexes; sequence-specific binding of p53 to DNA. (1) DNA looping in prokaryotes Stabilization of the multi-subunit protein-DNA complexes is facilitated by DNA looping, which brings the proteins close to each other. One of the best characterized is the gal loop in E. coli. To visualize such a complex structure, we used an innovative approach. First, genetic (sequence variability) data were used to determine the overall orientation of the gal repressors in a tetrameric structure. Next, the empirically based computer modeling developed by us earlier was employed to determine the optimal trajectory of the DNA loop. In particular, we found that the antiparallel gal loop is energetically more favorable than the parallel one. The same trend was found for the DNA loop formed upon binding of the lac repressors to DNA. The atomic force microscopy data support the antiparallel DNA looping, both with the galand lac repressors (in preparation). These results imply that the antiparallel DNA looping may be a general feature of the condensed bacterial nucleoid, as opposed to the parallel DNA wrapping around histones in eukaryotic chromatin. The hypothetical regular DNA folding in prokaryotes is consistent with the periodic distribution of the curved A-tracts in bacterial genomes, observed by us recently. Based on these findings, we designed detailed experiments to elucidate further the 3D organization of the DNA loops in bacteria (in collaboration with S. Adhya, NCI). (2) Elongation transcription complexes Another new method, enabling visualization of the DNA and RNA strands in the nucleoprotein complexes, is the iodine-125 radioprobing. This is based on analysis of the DNA strand breaks produced by the decay of an electron-emitting radioisotope, 125 I, incorporated in the C5 position of cytosine. The weaker the DNA break, the larger the distance from the radioisotope to the cleavage site. In this way, it is possible to reconstruct the 3D trajectories of nucleic acids in large nucleoprotein complexes. Using radioprobing, we detected for the first time the DNA-RNA heteroduplex outside the polymerase (in collaboration with R. Neumann and I. Panyutin, Clinical Center, NIH). Among other applications, this result suggests a novel mechanism for stabilization and regulation of the elongation complex. In addition to radioprobing and computer modeling, now we are studying the transcription complexes with time-resolved fluorescence, to visualize all the three DNA/RNA strands in real time. Our preliminary data imply different conformational behavior of the DNA strands during initiation and elongation (in collaboration with T. Jovin, MPI, Goettingen, Germany). We anticipate that this combined approach will give us a better understanding of those molecular mechanisms, which are responsible for the fidelity of RNA synthesis and are involved in recognition of the DNA mismatches in the course of transcription. (3) Sequence-specific p53-DNA binding The tetrameric p53-DNA complex plays the key role in tumor suppression. Therefore, it is important to elucidate the p53-DNA complex structure, as this could lead to understanding how formation of the tetrameric complex modifies binding sites on the p53 surface (which, in turn, serve as the signals for other members of the multi-protein cascade involved in the tumor supression pathway). Our early gel electrophoresis data revealed the DNA bending and increase in its twisting upon binding to p53, although the stereochemical details remained unclear. To determine the precise locations of the DNA bending and twisting in the tetrameric p53-DNA complex, we used the iodine-125 radioprobing described above. The DNA bending into the major groove was detected in the CATG tetramers in the centers of the two 10-mers (half-sites of the consensus 20-mer). Importantly, the internal CA:TG dimers (located closer to the center of the 20-mer) were distorted stronger than the external CA:TG (located closer to the ends of the p53 binding sites). This finding is consistent with the sequence variability observed for the known p53 response elements (in preparation). A next step is to analyze the effects of the p53 mutations and substitutions at the DNA response elements on the conformation and stability of the p53-DNA complex. Availability of the human DNA sequence allows determination of all potential p53 binding sites in the genome. We observed highly nonrandom distribution of such sites in all human chromosomes, reflecting the lateral p53 binding to the DNA loops, consistent with our electrophoresis measurements. We are currently exploring localization of the p53 sites with respect to the starts of transcription. The observed genome-wide distribution of the p53 sites also suggests further experiments regarding the p53 binding to nucleosomal DNA, and its possible role in gene regulation (in progress). The expansion of structural databases for nucleic acids, proteins and nucleoprotein complexes, as well as continued progress in elucidating the sequence-dependent structural properties of DNA and RNA, will improve the accuracy with which large nucleoprotein complexes can be characterized by the methods described above.

人类DNA测序的完成使人们对基因组功能的结构方面有了新的重视。在后基因组时代，了解基因调控的分子机制（通常通过蛋白质-核酸相互作用实现）变得更加重要。在大多数情况下，调节核蛋白复合物的结构分析需要使用非常规的方法，因为它们对于NMR和X射线晶体学来说太大了。下面介绍的项目旨在将我们最近开发的新方法应用于三个具体问题：原核阻遏体的组装;转录延伸复合物的稳定和调节; p53与DNA的序列特异性结合。 (1)原核生物中的DNA环 多亚基蛋白质-DNA复合物的稳定化通过DNA成环促进，这使蛋白质彼此靠近。最好的特征之一是大肠杆菌中的gal环。杆菌为了可视化如此复杂的结构，我们使用了一种创新的方法。首先，使用遗传（序列变异性）数据来确定gal阻遏物在四聚体结构中的总体方向。接下来，我们开发的基于经验的计算机建模被用来确定DNA环的最佳轨迹。特别是，我们发现，反平行半乳糖环是积极的比平行的更有利。在乳糖阻遏物与DNA结合后形成的DNA环中也发现了相同的趋势。原子力显微镜的数据支持反平行的DNA循环，无论是与galand乳糖阻遏物（准备）。 这些结果表明，反平行的DNA环可能是浓缩的细菌类核的一般特征，而不是平行的DNA包裹在真核染色质中的组蛋白周围。原核生物中DNA的规则折叠与我们最近观察到的细菌基因组中弯曲的A束的周期性分布是一致的。基于这些发现，我们设计了详细的实验来进一步阐明细菌中DNA环的3D组织（与S。Adhya，NCI）。 (2)延伸转录复合物 另一种新方法是碘-125放射性探测，它可以使核蛋白复合物中的DNA和RNA链可视化。这是基于分析的DNA链断裂所产生的衰变的电子发射放射性同位素，125 I，纳入C5位置的胞嘧啶。DNA断裂越弱，从放射性同位素到切割位点的距离越大。通过这种方式，可以重建大核蛋白复合物中核酸的3D轨迹。利用放射性探针，我们首次检测到聚合酶外的DNA-RNA异源双链体（与R。诺依曼和我。Panyeland，Clinical Center，NIH）。在其他应用中，这一结果表明了一种新的机制，用于稳定和调节的延伸复合物。 除了放射探测和计算机建模，现在我们正在研究具有时间分辨荧光的转录复合物，以真实的时间可视化所有三条DNA/RNA链。我们的初步数据暗示了DNA链在起始和延伸过程中的不同构象行为（与T。Jovin，MPI，Goettingen，德国）。我们预计，这种结合的方法将使我们更好地了解这些分子机制，这是负责的RNA合成的保真度，并参与识别的DNA错配在转录过程中。 (3)序列特异性p53-DNA结合 四聚体p53-DNA复合物在肿瘤抑制中起关键作用。因此，重要的是阐明p53-DNA复合物的结构，因为这可能导致理解四聚体复合物的形成如何改变p53表面上的结合位点（这反过来又充当参与肿瘤抑制途径的多蛋白级联的其他成员的信号）。我们早期的凝胶电泳数据显示，DNA弯曲和增加其扭曲后，结合p53，虽然立体化学的细节仍然不清楚。为了确定四聚体p53-DNA复合物中DNA弯曲和扭曲的精确位置，我们使用了上述碘-125放射性探测。 在两个10-mer的中心（共有20-mer的半位点）的CATG四聚体中检测到弯曲到大沟中的DNA。重要的是，内部CA：TG二聚体（位于更靠近20聚体的中心）比外部CA：TG（位于更靠近p53结合位点的末端）更强地扭曲。这一发现与已知p53反应元件（制备中）观察到的序列变异性一致。下一步是分析p53突变和DNA反应元件上的取代对p53-DNA复合物的构象和稳定性的影响。 人类DNA序列的可用性允许确定基因组中所有潜在的p53结合位点。我们观察到这些位点在所有人类染色体中的高度非随机分布，反映了p53与DNA环的横向结合，这与我们的电泳测量结果一致。我们目前正在探索的p53位点的转录开始的本地化。所观察到的p53位点的全基因组分布也表明了关于p53与核小体DNA结合的进一步实验，以及其在基因调控中的可能作用（正在进行中）。 核酸、蛋白质和核蛋白复合物的结构数据库的扩展，以及在阐明DNA和RNA的序列依赖性结构特性方面的持续进展，将提高通过上述方法表征大核蛋白复合物的准确性。