Biophysics of Macromolecular Complexes

大分子复合物的生物物理学

• 批准号: 7152481
• 负责人: rodolfo ghirlando
• 金额: --
• 依托单位: NATIONAL INSTITUTE OF DIABETES AND DIGESTIVE AND KIDNEY DISEASES
• 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/7152481
• 关键词: DNA repair; Escherichia coli; RNA; bacterial genetics; biophysics; chemical condensation; chemical structure; chemical structure function; chick embryo; chromatin; erythroid stem cell; folate; genetic transcription; globin; hemoprotein structure; intermolecular interaction; macromolecule; method development; nucleic acids; polymerase chain reaction; protein protein interaction; protein structure function; southern blotting; vitamin receptor

We are interested in the biophysical properties and structure of native chromatin fragments. The chicken folate receptor and beta-globin gene loci are ideal for such structural studies in that (i) the region possesses both condensed and transcriptionally active chromatin regions, and (ii) the system has been extensively studied in terms of gene regulation, allowing us to relate the overall chromatin structure to transcription. The condensed chromatin region, spanning 16 kbp of DNA flanked by the developmentally regulated folate receptor and beta-globin genes, can be released from the cell nucleus with the restriction enzyme HpaII. We have previously combined biophysical methods with real-time PCR and Southern blotting to analyze the hydrodynamic properties of this 16 kbp condensed chromatin and showed that it is an extended rod. This provides insights into the structure of heterochromatin, found interspersed within various genomes. We have furthered our studies of native chromatin fragments utilizing the methods we developed. Using an erythroid precursor cell line, we have analyzed a 16 kbp region of the transcriptionally poised beta-globin gene locus and showed that this chromatin fragment also appears to be an extended rod. However, unlike the condensed chromatin fragment, this chromatin region has a lower protein to nucleic acid ratio suggesting a role for RNA in the stabilization of this complex. Similar studies on 10-day old chicken erythrocyte chromatin, in which the beta-globin locus is transcriptionally active, are in progress. The persistence length (flexibility) of the chromatin fragments studied provides additional information and we are currently developing biochemical methods to determine the flexibility of such globin gene chromatin fragments. Macromolecular assemblies. In collaboration with members of the Laboratory of Molecular Biology, and other laboratories, protein and protein-nucleic acid assemblies have been characterized in terms of their shape, stoichiometry and affinity of interaction using hydrodynamic methods. These methods complement biochemical and structural investigations and provide important information on the biological mechanism. A case in point is provided by the recent studies of the conserved MutL protein carried out with Dr. Wei Yang. We showed that the C-terminal domain of MutL is a dimer. Furthermore, we showed that the proline-rich linker connecting the N- and C-termini is essentially rigid resulting in a greater than 10 nm end-to-end distance for the rod-shaped protein. Together with other data, these studies provide a working model for DNA mismatch repair in E. coli.

我们对天然染色质片段的生物物理性质和结构感兴趣。鸡叶酸受体和β -珠蛋白基因位点是这种结构研究的理想选择，因为(i)该区域具有浓缩和转录活性的染色质区域，（ii）该系统在基因调控方面已被广泛研究，使我们能够将整个染色质结构与转录联系起来。浓缩染色质区横跨16kbp的DNA，两侧是发育调节的叶酸受体和β -珠蛋白基因，可以通过限制性内切酶HpaII从细胞核中释放出来。我们之前将生物物理方法与实时PCR和Southern blotting相结合，分析了这个16 kbp浓缩染色质的流体动力学特性，并表明它是一个延长的棒。这提供了对异染色质结构的深入了解，异染色质分布在不同的基因组中。