Assembly architecture and DNA binding of the bacteriophage P22 terminase small subunit.
Assembly architecture and DNA binding of the bacteriophage P22 terminase small subunit.
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噬菌体P22末端酶小亚基的组装结构和DNA结合。
DOI：
10.1016/j.jmb.2008.08.050
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发表时间：
2008-11-14
影响因子：
5.6
通讯作者：
Thomas, George J., Jr.
中科院分区：
文献类型：
--
作者：
Nemecek, Daniel;Lander, Gabriel C.;Johnson, John E.;Casjens, Sherwood R.;Thomas, George J., Jr.
噬菌体P22的形态发生涉及将双链DNA包装成预组装的原衣壳。DNA的易位是由一种强大的病毒编码分子马达——端酶完成的，它由大亚基（gp2, 499个残基）和小亚基（gp3, 162个残基）组成。虽然gp2含有磷酸水解酶和末端酶的内切酶活性，但gp3的功能可能是调节特异性和非特异性的DNA识别模式以及gp2的酶活性。电镜显示野生型gp3自组装成稳定的单分散非美聚环。18 Å分辨率的三维重建提供了P22末端酶结构的第一次一瞥，并暗示了与DNA相互作用的两种不同模式-涉及20 Å直径的中心通道和34 Å分隔的径向尖峰。电迁移位移实验表明，gp3环在体外非特异性结合dsDNA，是通过gp3带正电的c端（残基143-152）和DNA主干的磷酸盐之间的静电相互作用。拉曼光谱显示，在残基142处截断的亚基形成的非美洲环保留了亚基折叠，尽管失去了dna结合活性。含有全长亚基和c端截断亚基的gp3环之间的密度差图与沿非美洲环中心通道的残基143-152的定位一致。结果提示gp3在DNA识别和易位中的功能可能的分子机制。
Morphogenesis of bacteriophage P22 involves the packaging of double-stranded DNA into a preassembled procapsid. DNA is translocated by a powerful virally-encoded molecular motor called terminase, which comprises large (gp2, 499 residues) and small (gp3, 162 residues) subunits. While gp2 contains the phosphohydrolase and endonuclease activities of terminase, the function of gp3 may be to regulate specific and nonspecific modes of DNA recognition as well as the enzymatic activities of gp2. Electron microscopy shows that wildtype gp3 self-assembles into a stable and monodisperse nonameric ring. A three-dimensional reconstruction at 18 Å resolution provides the first glimpse of P22 terminase architecture and implies two distinct modes of interaction with DNA – involving a central channel of 20 Å diameter and radial spikes separated by 34 Å. Electromobility shift assays indicate that the gp3 ring binds dsDNA nonspecifically in vitro via electrostatic interactions between the positively charged C-terminus of gp3 (residues 143–152) and phosphates of the DNA backbone. Raman spectra show that nonameric rings formed by subunits truncated at residue 142 retain the subunit fold, despite the loss of DNA-binding activity. Difference density maps between gp3 rings containing full-length and C-terminally truncated subunits are consistent with localization of residues 143–152 along the central channel of the nonameric ring. The results suggest a plausible molecular mechanism for gp3 function in DNA recognition and translocation.
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