Cooperative Assembly of Host and Viral Proteins into a DNA Packaging Motor Complex Analyzed by Fluorescence-Monitored Analytical Ultracentrifugation

Abstract

Packaging of viral genomes into procapsids by terminaseenzymes is conserved in complex double-stranded DNA viruses. Terminases bind to linear concatemers of replicated viral genomes linked end-to-end and concomitantly excise (mature) and translocate (package) a single genome per procapsid. While the terminase maturation complex requires site-specific recognition to mature the genome ends at each cos site within the concatemer, the terminase motor complex must bind DNA tightly, but non-specifically, during packaging. The bacteriophage λ protomer is a heterotrimer composed of two different subunits that separately confer catalytic activities and site-specific DNA binding. Although our data demonstrate that the protomer self-associates into a ring-like tetramer, the stoichiometries of terminase in the maturation and packaging complexes remains unknown. We investigated the nature of the terminase-DNA maturation complex and the factors that mediate site-specific vs. non-specific DNA binding modes. Specifically, we utilized electrophoretic mobility shift assays (EMSA) and analytical ultracentrifugation (AUC) to examine assembly of λ terminase and E. coli Integration Host Factor (IHF, required for λ development in vivo) on model DNA substrates. We first demonstrate that terminase protomer does not significantly discriminate between cos-containing and non-specific DNA substrates using EMSA; however, in the presence of IHF the enzyme forms specific complexes with cos-DNA at concentrations much lower than those required to bind non-specific DNA. We next utilize AUC to demonstrate that a binary complex is formed between cos-DNA and IHF. Addition of λ terminase to this binary complex affords two higher-order ternary complexes in a concentration-dependent manner. The data demonstrate that IHF promotes cooperative and site-specific assembly of terminase at the packaging initiation site cos. The nature of the nucleoprotein complexes will be discussed in relation to their role in viral genome packaging.