Dissecting Lambda Terminase: A Viral DNA Packaging Motor

Abstract

During virus assembly, a single genome is packaged into a preformed capsid by a terminase enzyme. Presently, the mechanistic details of the packaging motor are unknown. Single molecule experiments have demonstrated that viral DNA packaging motors are among the most powerful currently known, capable of packaging dsDNA to over 20 atmospheres within the capsid. Therefore, the mechanistic details of DNA packaging are of interest from both a biological (drug target), and engineering (nanomachine) standpoint. Bacteriophage Lambda (λ) is a model system for the study of dsDNA viruses, including herpesvirus and many bacteriophage. λ terminase is a multifunction enzyme complex with catalytic activities required to (1) recognize the viral genome, (2) prepare the viral genome for packaging, (3) recognize the empty procapsid, and (4) translocate viral DNA into the empty procapsid. The enzyme consists of two proteins, gpA and gpNu1 in a 1:2 ratio known as the heterotrimeric protomer; the motor functions as a tetramer of protomers assembled into a ring complex.Here, we study the λ terminase in vitro and characterize macromolecular assembly, genome packaging, ATPase and DNA-cleavage activities of several mutants which are catalytically deficient. These mutants include K76R, which is a mutation in the packaging ATPase site, T194M, which packages at a 10-fold slower rate in vivo, and G212S, which stalls during packaging. We characterize protomer self-assembly into the tetrameric motor and we compare the catalytic activities of the mutants relative to wild-type. The mechanistic implications of this work are discussed.