Measurements of single DNA molecule packaging dynamics in bacteriophage λ reveal high forces, high motor processivity, and capsid transformations
Measurements of single DNA molecule packaging dynamics in bacteriophage λ reveal high forces, high motor processivity, and capsid transformations
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DOI：
10.1016/j.jmb.2007.09.011
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发表时间：
2007-11-09
影响因子：
5.6
通讯作者：
Smith, Doucilas E.
中科院分区：
文献类型：
--
作者：
Fuller, Derek N.;Raymer, Dorian M.;Smith, Doucilas E.
在许多双链DNA病毒中，分子马达驱动基因组包装到预先形成的前衣壳中。在这里，我们提出了光镊测量单个DNA分子包装在噬菌体λ。将DNA-gpA-gpNu 1复合物与重组gpA和gpNu 1蛋白组装并栓系到微球上，并将原衣壳附着到单独的微球上。在ATP存在下，在将这些微球靠近的几秒钟内观察到DNA结合和包装的启动。观察到马达产生大于50皮牛顿（pN）的力，在与噬菌体phi 29相同的范围内，表明高力产生是病毒包装马达的共同特性。然而，在低衣壳填充时，包装速率平均类似于600 bp/s，这比phi 29高3.5倍，并且马达持续合成能力也高三倍，每个基因组长度易位少于一个滑动。包装速率随衣壳填充的增加而显著减慢，表明在86%包装时内力的积累达到14 pN，与在渗透压实验中测量的驱动DNA喷射的力和理论计算的一致。总之，这些实验表明，在包装过程中建立的内力在很大程度上可用于驱动随后的DNA喷射。此外，我们观察到在30%包装时平均包装速率下降80 bp/s，这表明在平均4 pN的外力积累后，在该点发生原衣壳扩增。在使用比野生型基因组更长的DNA构建体的实验中，在90%以上的包装中观察到包装速率的突然加速，并且远大于100%的基因组长度被易位，这表明内力可以使缺乏辅助蛋白（gpD）的未成熟的前衣壳破裂。(C)2007爱思唯尔有限公司保留所有权利。
Molecular motors drive genome packaging into preformed procapsids in many double-stranded (ds)DNA viruses. Here, we present optical tweezers measurements of single DNA molecule packaging in bacteriophage lambda. DNA-gpA-gpNu1 complexes were assembled with recombinant gpA and gpNu1 proteins and tethered to microspheres, and procapsids were attached to separate microspheres. DNA binding and initiation of packaging were observed within a few seconds of bringing these microspheres into proximity in the presence of ATP. The motor was observed to generate greater than 50 picoNewtons (pN) of force, in the same range as observed with bacteriophage phi 29, suggesting that high force generation is a common property of viral packaging motors. However, at low capsid filling the packaging rate averaged similar to 600 bp/s, which is 3.5-fold higher than phi 29, and the motor processivity was also threefold higher, with less than one slip per genome length translocated. The packaging rate slowed significantly with increasing capsid filling, indicating a buildup of internal force reaching 14 pN at 86% packaging, in good agreement with the force driving DNA ejection measured in osmotic pressure experiments and calculated theoretically. Taken together, these experiments show that the internal force that builds during packaging is largely available to drive subsequent DNA ejection. In addition, we observed an 80 bp/s dip in the average packaging rate at 30% packaging, suggesting that procapsid expansion occurs at this point following the buildup of an average of 4 pN of irternal force. In experiments with a DNA construct longer than the wild-type genome, a sudden acceleration in packaging rate was observed above 90% packaging, and much greater than 100% of the genome length was translocated, suggesting that internal force can rupture the immature procapsid, which lacks an accessory protein (gpD). (C) 2007 Elsevier Ltd. All rights reserved.