Garver K, Guo P
Department of Pathobiology, Purdue University, West Lafayette, Indiana 47907, USA.
J Biol Chem. 2000 Jan 28;275(4):2817-24. doi: 10.1074/jbc.275.4.2817.
During replication, the lengthy genome of double-stranded DNA viruses is translocated with remarkable velocity into a limited space within the procapsid. The question of how this fascinating task is accomplished has long been a puzzle. Our recent investigation suggests that phi29 DNA packaging is accomplished by a mechanism similar to the driving of a bolt with a hex nut and that six packaging RNAs (pRNAs) form a hexagonal complex to gear the DNA-translocating machine (Chen, C., and Guo, P. (1997) J. Virol. 71, 3864-3871; Zhang, F., Lemieux, S., Wu, X., St.-Arnaud, S., McMurray, C. T., Major, F., and Anderson, D. (1998) Mol. Cell 2, 141-147; Guo, P., Zhang, C., Chen, C., Garver, K., and Trottier, M., (1998) Mol. Cell 2, 149-155). In the current study, circularly permuted pRNAs were used to position an azidophenacyl photoreactive cross-linking agent specifically at a strategic site that was predicted to be involved in pRNA-pRNA interaction. Cross-linked pRNA dimers were isolated, and the sites of cross-link were mapped by primer extension. The cross-linked pRNA dimer retained full activity in phi29 procapsid binding and genomic DNA translocation, indicating that the cross-link distance constraints identified in dimer formation reflect the native pRNA complex. Both cross-linked dimers either containing or not containing the interlocking loops for programmed hexamer formation bound procapsid equally well; however, only the one containing the interlocking loops programmed for hexamer formation was active in phi29 DNA packaging. The cross-linked pRNA dimers were also identified as the minimum binding unit necessary for procapsid binding. Primer extension of the purified cross-linked pRNA dimers revealed that base G(82) was cross-linked to bases G(39), G(40), A(41), C(49), G(62), C(63), and C(64), which contribute to the formation of the three-way junction, suggesting that these bases are proximate in the formation of pRNA tertiary structure. Interestingly, the photoaffinity agent in the left interacting loop did not cross-link directly to the right loop as expected but cross-linked to bases adjacent to the right loop. These data provide a background for future modeling of pRNA tertiary structure.
在复制过程中，双链DNA病毒的长基因组以惊人的速度被转运到原衣壳内的有限空间中。长期以来，这个迷人的任务是如何完成的一直是个谜。我们最近的研究表明，φ29 DNA包装是通过一种类似于用六角螺母驱动螺栓的机制完成的，并且六个包装RNA（pRNA）形成一个六边形复合物来驱动DNA转运机器（Chen, C., and Guo, P. (1997) J. Virol. 71, 3864 - 3871; Zhang, F., Lemieux, S., Wu, X., St.-Arnaud, S., McMurray, C. T., Major, F., and Anderson, D. (1998) Mol. Cell 2, 141 - 147; Guo, P., Zhang, C., Chen, C., Garver, K., and Trottier, M., (1998) Mol. Cell 2, 149 - 155）。在当前的研究中，使用环状排列的pRNA将叠氮苯甲酰光反应性交联剂特异性地定位在一个预测参与pRNA - pRNA相互作用的关键位点。分离出交联的pRNA二聚体，并通过引物延伸法绘制交联位点。交联的pRNA二聚体在φ29原衣壳结合和基因组DNA转运中保留了全部活性，这表明在二聚体形成中确定的交联距离限制反映了天然的pRNA复合物。含有或不含有用于程序性六聚体形成的互锁环的两种交联二聚体与原衣壳的结合同样良好；然而，只有含有用于六聚体形成的互锁环的那种在φ29 DNA包装中具有活性。交联的pRNA二聚体也被确定为原衣壳结合所需的最小结合单元。对纯化的交联pRNA二聚体进行引物延伸显示，碱基G(82)与碱基G(39)、G(40)、A(41)、C(49)、G(62)、C(63)和C(64)交联在一起，并有助于形成三向连接，这表明这些碱基在pRNA三级结构形成中彼此靠近。有趣的是，左相互作用环中的光亲和剂并没有如预期那样直接与右环交联，而是与右环相邻的碱基交联。这些数据为未来pRNA三级结构的建模提供了背景。
