Single-Molecule Measurements of Motor-Driven Viral DNA Packaging in Bacteriophages Phi29, Lambda, and T4 with Optical Tweezers.
Single-Molecule Measurements of Motor-Driven Viral DNA Packaging in Bacteriophages Phi29, Lambda, and T4 with Optical Tweezers.
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使用光镊对噬菌体 Phi29、Lambda 和 T4 中电机驱动的病毒 DNA 包装进行单分子测量。
DOI：
10.1007/978-1-4939-8556-2_20
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发表时间：
2018
期刊：
影响因子：
--
通讯作者：
Smith,DouglasE
中科院分区：
文献类型：
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作者：
Keller,Nicholas;delToro,DamianJ;Smith,DouglasE
病毒DNA包装是许多dsDNA病毒组装中的必需步骤。由ATP水解提供燃料的分子马达在室温下约5分钟内将病毒基因组包装到预成型的prohead外壳内的接近晶体密度。我们描述的程序测量包装成单个病毒proheads与光镊的单个DNA分子。详细描述了三种病毒包装系统：噬菌体phi 29（φ29）、λ（λ）和T4。描述了两种不同的方法：（1）使用φ29和T4，可以批量预组装探针-马达复合物，并且可以通过将单个DNA分子“馈送”到其中一个复合物来在光镊中启动包装;（2）使用φ29和λ，可以批量启动包装然后停止，然后可以用光镊捕获单个探针-马达-DNA复合物并重新启动。在这两种情况下，探针头最终连接到一个捕获的微球上，被包装的DNA的末端连接到第二个捕获的微球上，使得DNA的包装将两个微球拉在一起，并且包装的速率和由马达产生的力在真实的时间内直接测量。这些协议允许许多实验参数对包装动力学的影响进行研究，如温度，ATP浓度，离子条件，DNA底物的结构变化，以及马达蛋白的突变。还描述了用光学陷阱捕获微球的程序和不同的测量模式。
Viral DNA packaging is a required step in the assembly of many dsDNA viruses. A molecular motor fueled by ATP hydrolysis packages the viral genome to near crystalline density inside a preformed prohead shell in ~5 min at room temperature. We describe procedures for measuring the packaging of single DNA molecules into single viral proheads with optical tweezers. Three viral packaging systems are described in detail: bacteriophages phi29 (φ29), lambda (λ), and T4. Two different approaches are described: (1) With φ29 and T4, prohead–motor complexes can be preassembled in bulk and packaging can be initiated in the optical tweezers by “feeding” a single DNA molecule to one of the complexes; (2) With φ29 and λ, packaging can be initiated in bulk then stalled, and a single prohead–motor–DNA complex can then be captured with optical tweezers and restarted. In both cases, the prohead is ultimately attached to one trapped microsphere and the end of the DNA being packaged is attached to a second trapped microsphere such that packaging of the DNA pulls the two microspheres together and the rate of packaging and force generated by the motor is directly measured in real time. These protocols allow for the effect of many experimental parameters on packaging dynamics to be studied such as temperature, ATP concentration, ionic conditions, structural changes to the DNA substrate, and mutations in the motor proteins. Procedures for capturing microspheres with the optical traps and different measurement modes are also described.