Motor-Substrate Interactions in a Ring ATPase

Abstract

Many processes in biology, including DNA recombination, prokaryotic cell-segregation, gene transposition, and viral DNA packaging, involve the translocation of DNA or RNA by ATP-driven ring motors belonging to the ASCE/AAA superfamily. While the mechanism by which these motors convert the chemical energy from ATP hydrolysis to mechanical work is beginning to be understood, little is known about how these motors engage their nucleic acid substrates. Do motors contact a single DNA element, such as a phosphate or a base, or are contacts distributed over multiple parts of the DNA? In addition, what role do these contacts play in the mechanochemical cycle? Here we use a single-molecule assay for the genome packaging motor of the Bacillus subtilis bacteriophage phi-29 to address these questions. The full mechanochemical cycle of the motor involves two phases--an ATP loading dwell followed by a translocation burst of four 2.5-bp steps. By challenging the motor with a variety of modified DNA substrates, we show that during the dwell phase important contacts are made with adjacent phosphates every 10-bp on the 5′-3′ strand in the direction of packaging. In addition to providing stable, long-lived contacts, these phosphate interactions also regulate the chemical cycle. In contrast, during the burst phase, we find that DNA translocation is driven against large forces by extensive contacts, some of which are not specific to the chemical moieties of DNA. Such promiscuous, non-specific contacts may reflect common translocase-substrate interactions for both the nucleic acid and protein translocases of the ASCE superfamily.