During coliphage lambda head morphogenesis two independent pathways converge. In one pathway, an isometric icosahedral protein shell (prohead) is assembled; in the other, concatemers of lambda chromosomes are synthesized as the product of DNA replication. Proheads and DNA are brought together in a very specific manner and a chromosome is inserted into the cavity of the prohead and separated from the remainder of the concatemer by cutting. The enzyme lambda DNA terminase, comprising the products of two lambda genes, - Nu1 and A - brings together DNA and proheads, thus serving as the recognition element for DNA packaging. The enzyme also generates the mature chromosomal ends by catalyzing the formation of the staggered nicks at cos, its specific duplex DNA target. In these activities, terminase is supported by another lambda-coded protein, the FI-gene product, and by one or more host protein factors (IHF, THF). The long term objective is to understand lambda DNA packaging in molecular detail. This involves the study of the structure and enzymatic transactions of terminase and the function of its supporting factors. This application deals mainly with the structure of the enzyme and of its complexes with DNA. For this purpose terminase will be purified in amounts of hundreds of milligrams to attempt its crystallization and, thus, the determination of its structure by X- ray diffractometry. To complete the study of the enzyme structure, the various domains of this enzyme (ATP- binding/hydrolysis, DNA-binding, nuclease, prohead binding) will be studied through the characterization of enzyme mutants, the cloning of DNA coding for enzyme fragments, the use of proteolysis (chemical, enzymatic) and photoaffinity labeling. The integration of these two sets of data will facilitate an understanding of how the enzyme works. Finally, an attempt will be made to determine the structure of the nucleoprotein particle that assembles during terminase binding to cos, by use of chemical cross-linking agents and two- dimensional polyacrylamide gel electrophoresis. This part of the study also extends to the role of the E. coli supporting factor (IHF) in the conformation of the nucleoprotein complex.
