STRUCTURE/FUNCTION OF PHI29 HEXAMERIC PRNA

蛋白质、核酸等大分子通过屏障或细胞膜的迁移或转位是生物系统中常见的过程。 其中最复杂和错综复杂的易位过程是病毒DNA的易位。所有线性双链DNA病毒，包括肝炎病毒、痘病毒、腺病毒和双链DNA噬菌体，都将其基因组DNA包装到预先形成的蛋白质外壳（原衣壳）中。 ATP被消耗，以提供能量，使冗长的基因组DNA以惊人的速度浓缩到原壳体内的有限空间中。 噬菌体phi 29需要病毒编码的120个碱基的RNA（pRNA）来包装其基因组DNA。 这种pRNA含有两个负责RNA间相互作用的单链环。 通过这种环/环相互作用，六个pRNA形成一个六聚体环，以驱动DNA包装机。我们假设pRNA具有至少两种功能构象。 由ATP水解驱动的pRNA六边形的每个成员的收缩和松弛之间的交替可以帮助旋转DNA运输机器。 我们的长期目标是阐明这种DNA运输马达是如何工作的，从而提供有关生物能量学以及蛋白质，DNA和RNA之间相互作用机制的基本信息。 本计划的短期目标是研究pRNA二聚体或六聚体的形成条件，并构建由六聚体pRNA复合物组成的六边形阵列，用于未来的结构分析。

Migration or translocation of macromolecules, such as proteins or nucleic acids, through barriers or cell membrane is a common process in biological system. One of the most complex and intricate translocation processes is viral DNA encapsidation. All linear dsDNA viruses, including hepesviruses, poxviruses, adenoviruses, and the ds-DNA bacteriophages package their genomic DNA into a preformed protein shell (procapsid). ATP is consumed to provide energy to condense the lengthy genomic DNA into the limited space inside procapsids with remarkable velocity. Phage phi29 requires a viral-encoded 120-base RNA (pRNA) to package its genomic DNA. This pRNA contains two single-stranded loops that are responsible for inter-RNA interactions. Through this loop/loop interaction, six pRNAs form a hexameric ring to gear the DNA packaging machine. We hypothesize that the pRNA possesses at least two functional conformations. Alternating between contraction and relaxation, driven by ATP hydrolysis, of each member of the pRNA hexagon could help rotate the DNA transportation machinery. Our long term objective is to elucidate how this DNA transportation motor works, thus providing fundamental information concerning bioenergetics as well as the mechanism of the interactions among protein, DNA and RNA. The short term objective of this proposal is to investigate the conditions for the formation of pRNA dimers or hexamers, and to construct hexagonal arrays composed of hexameric pRNA complex for future structural analysis.