Ring NTPases of the ASCE superfamily perform a variety of cellular functions. An important question about the operation of these molecular machines is how the ring subunits coordinate their chemical and mechanical transitions. Here, we present a comprehensive mechanochemical characterization of a homomeric ring ATPase - the bacteriophage 29 packaging motor - a homopentamer that translocates double-stranded DNA in cycles composed of alternating dwells and bursts. We use high-resolution optical tweezers to determine the effect of nucleotide analogs on the cycle. We find that ATP hydrolysis occurs sequentially during the burst and that ADP release is interlaced with ATP binding during the dwell, revealing a high degree of coordination among ring subunits. Moreover, we show that the motor displays an unexpected division of labor: although all subunits of the homopentamer bind and hydrolyze ATP during each cycle, only four participate in translocation, whereas the remaining subunit plays an ATP-dependent regulatory role.
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We thank M. Dangkulwanich, T. Ishibashi, C. Kaiser, N. Liu, R. Maillard, M. Morais, and M. Sen for critical discussions. This work was supported in part by grants from the National Institutes of Health (GM071552 to C.B., GM059604 and DE003606 to S.G.) and the U.S. Department of Energy, Office of Basic Energy Sciences, Division of Materials Sciences and Engineering under Contract No. DE-AC02-05CH11231 (single molecule force measurement) to C.B.