STRUCTURE/FUNCTION OF PHI29 HEXAMERIC PRNA

蛋白质或核酸等大分子通过屏障或细胞膜进行迁移或易位是生物系统中常见的过程。最复杂和复杂的易位过程之一是病毒DNA封装。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，都将它们的基因组DNA包裹在预先形成的蛋白质外壳（原衣壳）中。ATP的消耗是为了提供能量，以惊人的速度将冗长的基因组DNA浓缩到原壳体内有限的空间中。噬菌体phi29需要一种病毒编码的120碱基RNA （pRNA）来包装其基因组DNA。这个pRNA包含两个单链环，负责rna间的相互作用。通过这种环/环相互作用，六个prna形成一个六聚体环来驱动DNA包装机。我们假设pRNA至少具有两种功能构象。由ATP水解驱动的pRNA六边形的每个成员在收缩和松弛之间交替，可以帮助旋转DNA运输机制。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。本课题的短期目标是研究pRNA二聚体或六聚体形成的条件，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.