Nucleic acid enzymes studied at the molecular level

在分子水平上研究核酸酶

• 批准号: 6685308
• 负责人: STEVEN M BLOCK
• 金额: $ 30.79万
• 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 2005-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6685308
• 关键词: DNA; DNA directed RNA polymerase; Escherichia coli; RNA; active sites; adenosinetriphosphatase; biophysics; deoxyribopolynucleotide; endonuclease; enzyme mechanism; exonuclease; fluorescence; genetic transcription; helicase; interferometry; kinesin; mathematical model; model design /development; molecular dynamics; myosins; nanotechnology; nucleotidyltransferase; polyadenylate; protein transport; ribosomes

The Central Dogma of biology-whereby nucleic acids are replicated, transcribed, and translated into protein-is carried out by a core group of essential enzymes. These enzymes, which include polymerases, helicases, endo/exonucleases, and ribosomes, constitute a set of complex molecular machines. A detailed understanding of such machines is key to understanding life itself, and by extension to the treatment of disease. One property shared by these enzymes is that they function as processive motors: once bound to a DNA or RNA template, they carry out multiple enzymatic cycles. This motion is accompanied by the production of force and requires chemical energy, usually, but not always, in the form of nucleoside triphosphates (NTPs). In contrast to mechanoenzymes such as kinesin, the motor properties of nucleic acid enzymes are continually modulated by information in the template, yielding a rich dynamic behavior. Although high resolution structural data have become available for several nucleic acid enzymes, comparatively little is known about their mechanical properties and mechanisms. Recent work on biological motors has been revolutionized by the ability to monitor force and displacement at the single-molecule level, using biophysical instrumentation combined with in vitro motility assays. Beyond providing new ways of measurement, single molecule studies supply additional kinds of information- particularly about the distribution and heterogeneity of enzyme properties-that are not available with traditional biochemical methods, which yield ensemble averages. An assay for E. coli RNA polymerase now makes it possible to study transcription at the level of individual molecules, and prior work with this system has raised questions about elongation mechanisms, load- dependence, template specificity, pausing/stalling behavior, microstates, etc., that we plan to address through continuing study. We have also successfully developed a single-molecule assay for phage lambda exonuclease (a processive 5' yields 3' directed enzyme that degrades one strand of DNA), which we plan to exploit to learn more about its molecular mechanics, placing special emphasis on the load- and substrate-dependence. Unlike polymerases, or motors such as myosin and kinesin, lambda exonuclease is not powered by NTP hydrolysis, but by energy stored within the DNA itself. It will therefore be of long-term interest to compare and contrast newfound insights into the molecular mechanism of the exonuclease motor with what we learn about RNA polymerase through similar, single-molecule approaches.

生物学的中心教条 - 核酸被复制，转录并翻译成蛋白质，由一组必需酶进行。 这些酶，包括聚合酶，解旋酶，内核酸酶和核糖体构成一组复杂的分子机。 对此类机器的详细理解是理解生命本身以及扩展疾病治疗的关键。 这些酶共有的一个特性是它们充当过程中的电动机：一旦与DNA或RNA模板结合，它们就进行了多个酶促循环。 这种运动伴随着力的产生，通常需要化学能，但并非总是以三磷酸核苷（NTPS）的形式。与机械酶（例如驱动蛋白）相反，核酸酶的运动特性不断受模板中的信息调节，从而产生了丰富的动态行为。尽管高分辨率的结构数据已用于几种核酸酶，但对其机械性能和机制的了解相对较少。 使用生物物理仪器结合体外运动测定法，对生物电动机的最新工作已经通过在单分子水平监测力和位移的能力进行了革新。 除了提供新的测量方法外，单分子研究还提供了其他类型的信息 - 尤其是有关酶特性的分布和异质性的信息 - 传统的生化方法无法获得，从而产生集成平均值。 现在，对大肠杆菌RNA聚合酶的测定方法可以在单个分子水平上研究转录，并且先前与该系统的工作提出了有关伸长机制，负载依赖性，模板特异性，暂停/失速行为，微层等的问题，我们计划通过连续研究来解决。 我们还成功地开发了一种用于噬菌体lambda外切酶的单分子测定法（造成5'的惯用酶3'定向酶，使一链DNA降解），我们计划利用它更多地了解其分子力学，将其特殊强调放在负载和副物质依赖性上。 与聚合酶或诸如肌球蛋白和驱动蛋白等电机不同，Lambda核酸酶不是由NTP水解动力，而是由存储在DNA本身中的能量。 因此，将新鲜核酸酶电动机的分子机制与我们通过相似的单分子方法进行比较和对比的新发现的见解与对RNA聚合酶的知识将是长期的关注。