Nucleic Acid Enzymes Studied at the Molecular Level

分子水平上的核酸酶研究

• 批准号: 7152900
• 负责人: STEVEN M BLOCK
• 金额: $ 49.85万
• 依托单位: STANFORD UNIVERSITY
• 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 2009-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7152900
• 关键词: Address; Algorithms; Atomic Force Microscopy; Bacteriophages; Behavior; Binding; Binding Proteins; Biochemical; Biochemical Pathway; Biological Assay; Biology; Cations; Characteristics; Chemicals; Code; Complement; Complex; Condition; Coupled; Cyclobutanes; DNA; DNA Sequence; DNA-Directed RNA Polymerase; Data; Dependence; Development; Devices; Dissociation; Engineering; Enzymes; Escherichia coli; Event; Exonuclease; Fluorescence; Generations; Genetic; Genetic Transcription; Goals; Grant; Heterogeneity; Hour; Human; In Vitro; Individual; Kinesin; Laboratories; Lasers; Life; Measurement; Measures; Messenger RNA; Methods; Modeling; Molecular; Molecular Motors; Motion; Motor; Motor Neurons; Movement; Muscle; Myosin ATPase; Nanotechnology; Nature; Noise; Nucleic Acids; Optics; Organelles; Phase; Photons; Polymerase; Positioning Attribute; Production; Progress Reports; Property; Proteins; RNA; RNA Folding; Range; Recombinants; Records; Regulation; Research; Research Personnel; Resolution; Ribosomes; Role; Rotation; Score; Signal Transduction; Specificity; System; Techniques; Testing; Thinking; Thymidine; Time; Torque; Transcriptional Regulation; Translating; Work; antitermination factor; base; design; dimer; helicase; human disease; improved; instrument; instrumentation; laser tweezer; molecular scale; nano; nanoscale; novel; nuclease; nucleoside triphosphate; optical traps; programs; repaired; research study; single molecule; size; theories

DESCRIPTION (provided by applicant): The Central Dogma of biology-whereby DNA is replicated, transcribed, and ultimately translated into protein-is carried out by a handful of important enzymes. These enzymes, which include polymerases, helicases, nucleases, and ribosomes, constitute a core group of sophisticated, biomolecular machines. A detailed understanding of such machines is key to any understanding of life itself, and by extension, to the treatment of human disease. Furthermore, progress towards revealing the molecular mechanisms that power Nature's own molecular-scale machines is directly informing much of our current work at the forefront of nanotechnology, which promises to harness the power of nanoscale devices for the betterment of the human condition. Put simply, we need to know how these machines work if we're to fix them-or to copy them. A property shared by many nucleic acid enzymes is that they function as processive motors: once bound to their DNA template, they carry out repeated enzymatic cycles, often moving long distances before detaching. This motion is accompanied by the production of force, and requires a continuous input of chemical energy, usually in the form of nucleoside triphosphates. In contrast to classical mechanoenzymes like myosin (which moves muscles) or kinesin (which transports organelles in cells), the motor-like properties of nucleic acid enzymes are continually modulated by the changing information found in the DNA template, yielding a much richer dynamic behavior. Although high-resolution structural data have become available for many important nucleic acid enzymes, comparatively little is understood about the underlying molecular mechanisms. Recently, work on molecular motors has been revolutionized by the ability to measure force and displacement at the level of single molecules, using a new generation of biophysical instrumentation, including laser-based optical traps, scanning force microscopy, and advanced fluorescence techniques that can score single photons. Single-molecule studies hold great promise because they supply unique information-particularly about the distribution and heterogeneity of enzymatic properties-that's been largely inaccessible using traditional biochemical or genetic approaches. An assay developed by my group has allowed us to study gene transcription by E. coli RNA polymerase (RNAP) in real time at the level of individual molecules using optical traps. Our prior work with this system has raised specific questions about the elongation mechanism, the load-dependence, the DNA sequence specificity, pausing & stalling behavior, enzyme microstates, enzyme regulation, repair mechanisms, etc., that we're now in an excellent position to address through continuing study. Single-molecule techniques may even become sensitive enough to measure the size of the individual steps taken by RNAP, which are expected to correspond directly to the spacing of individual bases along the DNA (3.4Angstroms). A direct demonstration that RNAP advances in single basepair increments would allow us to rule out a competing theory that it moves by a so-called "inchworm" mechanism.

描述（由申请人提供）：生物学的中心教条 - DNA的重复，转录并最终被少数重要的酶进行的蛋白质IS。这些酶，包括聚合酶，解旋酶，核酸酶和核糖体，构成了复杂的生物分子机器的核心组。对此类机器的详细理解是对生命本身的任何理解，并扩展到治疗人类疾病的关键。此外，朝着揭示Power自然界自己的分子规模机器的分子机制的进展直接向我们目前的大部分工作介绍了纳米技术的最前沿，该机构有望利用纳米级设备的力量来改善人类状况。简而言之，如果我们要修复它们或复制它们，我们需要知道这些机器的工作原理。许多核酸酶共有的特性是它们充当过程中的电机：一旦与DNA模板结合，它们就会进行重复的酶促循环，通常在脱离之前移动长距离。这种运动伴随着力的产生，通常需要以核苷三磷酸盐形式进行化学能的连续输入。与经典的机械酶（如肌球蛋白（移动肌肉）或驱动蛋白（在细胞中运输细胞器））相反，核酸酶的运动性特性不断受到DNA模板中不断变化的信息的调节，从而产生了更丰富的动力学行为。尽管高分辨率的结构数据已用于许多重要的核酸酶，但对基本分子机制的了解相对较少。最近，使用新一代生物物理仪器（包括基于激光的光学诱捕器，扫描力显微镜和高级荧光技术），在单分子水平上测量力和位移的能力进行了革命性的革新。单分子研究具有很大的希望，因为它们提供了有关酶特性的分布和异质性的独特信息 - 使用传统的生化或遗传方法，这种酶特性的分布和异质性很大程度上是无法访问的。我组开发的一种测定方法使我们能够使用光学陷阱在单个分子水平上实时研究大肠杆菌RNA聚合酶（RNAP）的基因转录。我们先前与该系统的工作提出了有关伸长机制，载荷依赖性，DNA序列特异性，暂停和失速行为，酶微骨，酶调节，修复机制等的特定问题，我们现在可以通过继续研究来解决一个良好的位置。单分子技术甚至可能变得足够敏感，可以测量RNAP采取的单个步骤的大小，这有望直接与沿DNA沿DNA（3.4angstroms）的单个碱基的间距相对应。直接证明RNAP在单基台上增量中的进步将使我们排除竞争理论，即它通过所谓的“ Inchworm”机制移动。