Biophysics of Macromolecular Complexes

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-珠蛋白基因位点是理想的结构研究，因为（i）该区域具有浓缩和转录活性的染色质区域，以及（ii）该系统已被广泛研究的基因调控，使我们能够将整体染色质结构与转录。凝聚的染色质区域，跨越16 kbp的DNA两侧发育调控叶酸受体和β-珠蛋白基因，可以从细胞核释放的限制酶HpaII。我们以前结合生物物理方法与实时PCR和Southern印迹分析的流体动力学特性，这16 kbp的浓缩染色质，并表明它是一个延长杆。这提供了异染色质结构的见解，发现散布在各种基因组中。 我们利用我们开发的方法进一步研究了天然染色质片段。使用红系前体细胞系，我们已经分析了16 kbp的区域的转录平衡β-珠蛋白基因座，并表明，该染色质片段似乎也是一个延长杆。然而，与浓缩的染色质片段不同，该染色质区域具有较低的蛋白质与核酸的比率，表明RNA在该复合物的稳定中的作用。对10天大的鸡红细胞染色质的类似研究正在进行中，其中β-珠蛋白位点是转录活性的。染色质片段的持久性长度（灵活性）的研究提供了额外的信息，我们目前正在开发的生物化学方法，以确定这种珠蛋白基因染色质片段的灵活性。 大分子组装体。 与分子生物学实验室和其他实验室的成员合作，蛋白质和蛋白质-核酸组装体的特征在于它们的形状，化学计量和相互作用的亲和力。这些方法补充了生物化学和结构研究，并提供了生物学机制的重要信息。一个恰当的例子是最近与杨伟博士一起对保守MutL蛋白进行的研究。我们发现MutL的C-末端结构域是二聚体。此外，我们发现连接N-和C-末端的富含脯氨酸的接头基本上是刚性的，导致棒状蛋白质的端到端距离大于10 nm。结合其他数据，这些研究为大肠杆菌DNA错配修复提供了一个工作模型。杆菌

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.