SUMMARY Ring NTPases are a class of ubiquitous molecular motors involved in basic biological partitioning processes. dsDNA viruses encode ring ATPases that translocate their genomes to near-crystalline densities within pre-assembled viral capsids. Here, X-ray crystallography, cryoEM, and biochemical analyses of the dsDNA packaging motor in bacteriophage phi29 show how individual subunits are arranged in a pentameric ATPase ring, and suggest how their activities are coordinated to translocate dsDNA. The resulting pseudo-atomic structure of the motor and accompanying functional analyses show how ATP is bound in the ATPase active site; identify two DNA contacts, including a potential DNA translocating loop; demonstrate that a trans-acting arginine finger is involved in coordinating hydrolysis around the ring; and suggest a functional coupling between the arginine finger and the DNA translocating loop. The ability to visualize the motor in action illuminates how the different motor components interact with each other and with their DNA substrate.

Double-stranded DNA bacteriophages and their eukaryotic virus counterparts have twelve-fold head-tail connector assemblages embedded at a unique capsid vertex. This vertex is the site of assembly of the DNA packaging motor, and the connector has a central channel through which viral DNA passes during genome packaging and subsequent host infection. Crystal structures of connectors from different phages reveal either disordered residues or structured loops that project into the connector channel. Given the proximity to the translocating DNA substrate, these loops have been proposed to play a role in DNA packaging. Previous models have proposed structural motions in either the packaging ATPase or the connector channel loops as the driving force that translocates the DNA into the prohead. Here we mutate the channel loops of the Bacillus subtilis bacteriophage φ29 connector and show that these loops have no active role in translocation of DNA. Instead, they appear to have an essential function near the end of packaging, acting to retain the packaged DNA in the head in preparation for motor detachment and subsequent tail assembly and virion completion.

Subunits in multimeric ring-shaped motors must coordinate their activities to ensure correct and efficient performance of their mechanical tasks. Here, we study WT and arginine finger mutants of the pentameric bacteriophage φ29 DNA packaging motor. Our results reveal the molecular interactions necessary for the coordination of ADP-ATP exchange and ATP hydrolysis of the motor's biphasic mechanochemical cycle. We show that two distinct regulatory mechanisms determine this coordination. In the first mechanism, the DNA up-regulates a single subunit's catalytic activity, transforming it into a global regulator that initiates the nucleotide exchange phase and the hydrolysis phase. In the second, an arginine finger in each subunit promotes ADP-ATP exchange and ATP hydrolysis of its neighbor. Accordingly, we suggest that the subunits perform the roles described for GDP exchange factors and GTPase-activating proteins observed in small GTPases. We propose that these mechanisms are fundamental to intersubunit coordination and are likely present in other ring ATPases.

The DNA packaging motor of the Bacillus subtilis bacteriophage ø29 prohead is comprised in part of an oligomeric ring of 174-base RNA molecules (pRNA) positioned near the N-termini of subunits of the dodecameric head-tail connector. Deletion and alanine substitution mutants in the connector protein (gp10) N-terminus were assembled into proheads in Escherichia coli and the particles tested for pRNA binding and DNA-gp3 packaging in vitro. The basic amino acid residues RKR at positions 3-5 of the gp10 N-terminus were central to pRNA binding during assembly of an active DNA packaging motor. Conjugation of Fe-BABE to residue S170C in the narrow end of the connector, near the N-terminus, permitted hydroxyl radical probing of bound [ 32 P]pRNA and identified two discrete sites proximal to this residue: the C-helix at the junction of the A, C and D helices, and the E helix and the CE loop/D loop of the intermolecular base pairing site.

covery of the disease (Ross and Brim, 1957) and was quickly incorporated into breeding programs. Resis-Transgenic hairy roots of soybean [Glycine max (L.) Merrill] intance to SCN is an oligogenic quantitative trait (Anand duced by Agrobacterium rhizogenes support the complete life cycle of soybean cyst nematode (SCN, Heterodera glycines Ichinohe) inand Rao-Arelli, 1989;Young, 1996). Restriction fragvitro. However, expression of SCN resistance in hairy soybean roots ment length polymorphism (RFLP) mapping and linkhas not been investigated. A transgenic hairy root system would be age analyses have shown that at least three quantitative useful in developing an assay for candidate SCN resistance genes.Agric. Exp. Stn. This work was supported in part by USDA/95-37300-1593.