Department of Biochemistry and Molecular Biology, University of Maryland School of Medicine, Baltimore, MD 21201, USA.
Proc Natl Acad Sci U S A. 2012 Dec 11;109(50):20419-24. doi: 10.1073/pnas.1214318109. Epub 2012 Nov 26.
Viral genome packaging into capsids is powered by high-force-generating motor proteins. In the presence of all packaging components, ATP-powered translocation in vitro expels all detectable tightly bound YOYO-1 dye from packaged short dsDNA substrates and removes all aminoacridine dye from packaged genomic DNA in vivo. In contrast, in the absence of packaging, the purified T4 packaging ATPase alone can only remove up to ∼1/3 of DNA-bound intercalating YOYO-1 dye molecules in the presence of ATP or ATP-γ-S. In sufficient concentration, intercalating dyes arrest packaging, but rare terminase mutations confer resistance. These distant mutations are highly interdependent in acquiring function and resistance and likely mark motor contact points with the translocating DNA. In stalled Y-DNAs, FRET has shown a decrease in distance from the phage T4 terminase C terminus to portal consistent with a linear motor, and in the Y-stem DNA compression between closely positioned dye pairs. Taken together with prior FRET studies of conformational changes in stalled Y-DNAs, removal of intercalating compounds by the packaging motor demonstrates conformational change in DNA during normal translocation at low packaging resistance and supports a proposed linear "DNA crunching" or torsional compression motor mechanism involving a transient grip-and-release structural change in B form DNA.
病毒基因组包装成衣壳是由高力产生的马达蛋白驱动的。在存在所有包装成分的情况下，体外 ATP 驱动的易位将所有可检测到的紧密结合的 YOYO-1 染料从包装的短 dsDNA 底物中排出，并将体内包装的基因组 DNA 中的所有吖啶染料去除。相比之下，在不存在包装的情况下，纯化的 T4 包装 ATP 酶单独在 ATP 或 ATP-γ-S 的存在下只能去除多达约 1/3 的 DNA 结合的嵌入 YOYO-1 染料分子。在足够的浓度下，嵌入染料会阻止包装，但罕见的末端酶突变赋予了抗性。这些遥远的突变在获得功能和抗性方面高度相互依赖，可能标志着与正在移位的 DNA 接触的马达接触点。在停滞的 Y-DNAs 中，FRET 显示噬菌体 T4 末端酶 C 末端到入口的距离减小，与线性马达一致，并且在紧密定位的染料对之间的 Y 型 DNA 压缩中。与停滞的 Y-DNAs 中构象变化的先前 FRET 研究相结合，包装马达去除嵌入化合物证明了在低包装阻力下正常易位过程中 DNA 的构象变化，并支持了一种线性“DNA 压碎”或扭转压缩马达机制的提议，该机制涉及 B 型 DNA 中的瞬时握持和释放结构变化。
