By use of a model system that has several advantageous properties, we propose to investigate the mechanisms by which macromolecules cooperate to achieve a biologically evolved purpose. The model system consists of the components required during the packaging of double-stranded DNA in the DNA-free procapsid of bacteriophage T7. The advantageous properties include accessibility to the following procedures that we have developed for this type of investigation: first, analytical procedures for detecting, characterizing and quantifying particles in intermediate states of packaging (DNA packaging intermediates), and second, a high efficiency (more than 20%) in vitro procedure for producing the end product. In past studies, we h ave analyzed several DNA packaging intermediates, including capsids with incompletely packaged DNA. The data yield a model for both the sequence of events at the beginning of packaging and the mechanism for transduction of energy during entry of DNA into a capsid.
The specific aims are the following: (a) By use of both ultracentrifugation and our unusually capable, newly-developed techniques of nondenaturing gel electrophoresis, we will both detect and characterize additional T7 DNA packaging intermediates. Our current model for the DNA packaging pathway will be both tested and, if correct, extended. (b) We will test models for the mechanism of energy transduction during in vitro T7 DNA packaging. For this purpose, video light microscopy will be performed of single DNA molecules being packaged. (c) To assist in comparing in vitro to in vivo results, we will investigate the effects on in vitro DNA packaging of nonspecific factors present in vivo, but not yet accurately mimicked in vitro. Thes four include excluded volume, lowered water activity and the presence of a sieving network. (d) To assist in achieving specific aims (a) and (b) we will investigate the effects of in vitro packaging of removing molecules that are present in vivo, but are not necessary for producing the end product. We will develop an in vitro system of purified components that mimics in vivo DNA packaging. The proposed work will answer questions of general significance concerning biochemical pathways biological energy transduction and the relationship of in vitro to in vivo systems. Answering of these questions will open new approaches to antiviral therapies. This proposal is interdisciplinary.
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