DNA GEOMETRY IN TATA BINDING PROTEIN DNA COMPLEXES

TATA 结合蛋白 DNA 复合物中的 DNA 几何结构

• 批准号: 2771034
• 负责人: JASON D KAHN
• 金额: $ 9.51万
• 依托单位: UNIV OF MARYLAND, COLLEGE PARK
• 依托单位国家: 美国
• 财政年份: 1997
• 资助国家: 美国
• 起止时间: 1997-09-30 至 2000-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2771034
• 关键词: DNA binding protein; DNA footprinting; X ray crystallography; computer simulation; genetic promoter element; genetic regulation; genetic transcription; protein structure; transcription factor

DESCRIPTION: TATA-binding protein (TBP) is the DNA-binding component of transcription factor IID and recognizes the TATA box sequence in the promoter region of eukaryotic genes. The x-ray co-crystal structure of TBP bound to DNA shows that the protein induces a large bend in the DNA. The proposed research is aimed at determining the size and direction of the bend when TBP binds to DNA in solution, in order to compare this information with the crystal structure. Methodologies center on assays based on DNA ring closure and high resolution foot printing. The interpretation of experimental results will be aided by Monte Carlo computer simulation methods. It is proposed that comparison of the observed degree of bending in the crystal and in solution with the level of transcription for different promoters will allow the assessment of the functional importance of DNA bending, both in TBP binding, and in the regulation of transcription. The motivation for the work is the X-ray structure of TBP bound to DNA, which shows that the protein induces a large degree of bending and unwinding in the DNA upon binding. This bending is important in transcription initiation, in that the bend can alter the appearance of the DNA and thereby potentiate or prevent binding by other factors (e.g. TFIEB), or bending can bring together other proteins that would otherwise be separated on the DNA. This project concerns the DNA bending and unwinding in solution induced by TBP and TBP-containing multi-protein complexes. The first goal is the determination of the size and direction of the bend induced by TBP at a wild type TATA box and several variants, using quantitative DNA ring closure and minicircle binding. DNA molecules containing a TATA box and an intrinsic DNA bend have been constructed. When the TBP bend and the intrinsic bend cooperate to form the DNA into a C-shape, the DNA cyclizes very rapidly, whereas when the bends form an "S"-shape then cyclization is very slow. This semiqualtitative information is made more quantitative through Monte Carlo simulation. Through a collaboration with the Burley laboratory, comparison of the observed degree of bending in the crystal and in solution with the level of transcription supported by the mutant promoters will enable assessment of the functional importance of DNA bending, both in the process of TBP binding and in the overall process of transcription. Further, less well documented aims utilize similar techniques to study DNA bending as other factors, activators, and repressors (e.g. TFIIB, NC2) bind to the TBP-DNA complex.

描述：塔塔结合蛋白（TBP）是dna结合成分