biostudies-literatureDai LNIGMS NIH HHSNational Science Foundation6548https://www.ebi.ac.uk/biostudies/studies/S-EPMC8589836biostudies-literatureUnknown12(1)Multi-subunit ring-ATPases carry out a myriad of biological functions, including genome packaging in viruses. Though the basic structures and functions of these motors have been well-established, the mechanisms of ATPase firing and motor coordination are poorly understood. Here, using single-molecule fluorescence, we determine that the active bacteriophage T4 DNA packaging motor consists of five subunits of gp17. By systematically doping motors with an ATPase-defective subunit and selecting single motors containing a precise number of active or inactive subunits, we find that the packaging motor can tolerate an inactive subunit. However, motors containing one or more inactive subunits exhibit fewer DNA engagements, a higher failure rate in encapsidation, reduced packaging velocity, and increased pausing. These findings suggest a DNA packaging model in which the motor, by re-adjusting its grip on DNA, can skip an inactive subunit and resume DNA translocation, suggesting that strict coordination amongst motor subunits of packaging motors is not crucial for function.Nature communicationsA viral genome packaging ring-ATPase is a flexibly coordinated pentamer.PMC8589836PHY 1430124R35 GM122569R01 GM118817Chemla YRVafabakhsh RDai LKottadiel VILu SMahalingam MRao VBSingh DHa TfalseA viral genome packaging ring-ATPase is a flexibly coordinated pentamer.Multi-subunit ring-ATPases carry out a myriad of biological functions, including genome packaging in viruses. Though the basic structures and functions of these motors have been well-established, the mechanisms of ATPase firing and motor coordination are poorly understood. Here, using single-molecule fluorescence, we determine that the active bacteriophage T4 DNA packaging motor consists of five subunits of gp17. By systematically doping motors with an ATPase-defective subunit and selecting single motors containing a precise number of active or inactive subunits, we find that the packaging motor can tolerate an inactive subunit. However, motors containing one or more inactive subunits exhibit fewer DNA engagements, a higher failure rate in encapsidation, reduced packaging velocity, and increased pausing. These findings suggest a DNA packaging model in which the motor, by re-adjusting its grip on DNA, can skip an inactive subunit and resume DNA translocation, suggesting that strict coordination amongst motor subunits of packaging motors is not crucial for function.2021-01-01T00:00:00Z2021 Nov2024-02-15T19:06:59.291Z2022-02-11T12:53:48.249ZS-EPMC85898363477293610.1038/s41467-021-26800-z