Rawson Brandon, Yang Qin, Catalano Carlos E, Smith Douglas E
Department of Physics, University of California, San Diego, La Jolla, CA, 92093, USA.
Department of Pharmaceutical Sciences, Skaggs School of Pharmacy and Pharmaceutical Sciences, University of Colorado Denver, Anschutz Medical Campus, Aurora, CO, 80045, USA.
Sci Rep. 2025 Feb 27;15(1):7093. doi: 10.1038/s41598-024-74915-2.
Biomotor-driven DNA packaging is a key step in the life cycle of many viruses. We previously developed single-molecule methods using optical tweezers to measure packaging dynamics of the bacteriophage lambda motor. The lambda system is more complex than others examined via single-molecule assays with respect to the packaging substrate and ancillary proteins required. Because of this, previous studies which efficiently detected packaging events used crude E. coli cell extracts containing host factors and the terminase packaging enzyme. However, use of extracts is suboptimal for biochemical manipulation and obfuscates interrogation of additional factors that affect the process. Here we describe an optical tweezers assay using purified lambda terminase holoenzyme. Packaging events are as efficient as with crude extracts, but only if purified E. coli integration host factor (IHF) is included in the motor assembly reactions. We find that the ATP-driven DNA translocation dynamics, motor force generation, and motor-DNA interactions without nucleotide are virtually identical to those measured with extracts. Thus, single-molecule packaging activity can be fully recapitulated in a minimal system containing only purified lambda procapsids, purified terminase, IHF, and ATP. This sets the stage for single-molecule studies to investigate additional phage proteins known to play essential roles in the packaging reaction.
生物马达驱动的DNA包装是许多病毒生命周期中的关键步骤。我们之前开发了使用光镊的单分子方法来测量噬菌体λ马达的包装动力学。就所需的包装底物和辅助蛋白而言，λ系统比通过单分子测定法研究的其他系统更为复杂。因此，之前有效检测包装事件的研究使用了含有宿主因子和末端酶包装酶的大肠杆菌粗提物。然而，使用粗提物进行生化操作并不理想，并且会模糊对影响该过程的其他因素的探究。在此，我们描述了一种使用纯化的λ末端酶全酶的光镊测定法。包装事件与使用粗提物时一样高效，但前提是在马达组装反应中包含纯化的大肠杆菌整合宿主因子（IHF）。我们发现，由ATP驱动的DNA转位动力学、马达力的产生以及无核苷酸时的马达与DNA相互作用，实际上与使用粗提物测量的结果相同。因此，在仅包含纯化的λ原衣壳、纯化的末端酶、IHF和ATP的最小系统中，可以完全重现单分子包装活性。这为单分子研究奠定了基础，以研究已知在包装反应中起关键作用的其他噬菌体蛋白。
