Superstructure of the centromeric complex of TubZR C plasmid partitioning systems

Abstract

Bacterial plasmid partitioning systems segregate plasmids into each daughter cell. In the well-understood ParMRC plasmid partitioning system, adapter protein ParR binds to centromere parC, forming a helix around which the DNA is externally wrapped. This complex stabilizes the growth of a filament of actin-like ParM protein, which pushes the plasmids to the poles. The TubZRC plasmid partitioning system consists of two proteins, tubulin-like TubZ and TubR, and a DNA centromere, tubC, which perform analogous roles to those in ParMRC, despite being unrelated in sequence and structure. We have dissected in detail the binding sites that comprise Bacillus thuringiensis tubC, visualized the TubRC complex by electron microscopy, and determined a crystal structure of TubR bound to the tubC repeat. We show that the TubRC complex takes the form of a flexible DNA-protein filament, formed by lateral coating along the plasmid from tubC, the full length of which is required for the successful in vitro stabilization of TubZ filaments. We also show that TubR from Bacillus megaterium forms a helical superstructure resembling that of ParR. We suggest that the TubRC DNA-protein filament may bind to, and stabilize, the TubZ filament by forming such a ring-like structure around it. The helical superstructure of this TubRC may indicate convergent evolution between the actincontaining ParMRC and tubulin-containing TubZRC systems.FtsZ | X-ray crystallography | DNA segregation L ow copy number plasmids often encode their own segregation machinery, ensuring that copies are partitioned into each daughter cell. These plasmid-partitioning systems organize replicated plasmids and actively separate them. The known plasmidpartitioning systems are minimalist and elegant. They consist of just three components: a DNA centromere, an adapter protein that binds the centromere, forming the centromeric complex, and a nucleotide triphosphate-dependent filament-forming protein, which produces the force to move the plasmids (1, 2).Plasmid partitioning systems have been divided into types I-III, based upon the homology of their filament-forming proteins to known protein families (2, 3). Type I plasmid partitioning systems (4, 5) are based on deviant Walker A ATPases (6, 7), type II (8) on actin-like proteins (9), and type III (10-12) on tubulin/FtsZ-like proteins (13,14). Although the structure of the filament implicated in segregation has been determined for all three systems (9, 14-16), only examples of type I and II centromeric complexes have so far been resolved (17)(18)(19).The centromeric complex of the type II (actin-like) ParMRC plasmid partitioning system is formed by the binding of adapter protein ParR to parC, a series of parallel (direct), 11-base pair (bp) repeats (20). The superstructure this complex forms is helical, right-handed, and places the parC DNA on the exterior of the helix and ParR on the interior (18,19). This arrangement clusters the interaction surfaces between ParM and ParR, which are located at the tip of the ParM filament and ...

Reconstitution of a prokaryotic minus end-tracking system using TubRC centromeric complexes and tubulin-like protein TubZ filaments

“…Note that some background intensity fluctuations were caused by a small operator's light near the microscope. (6,17). Highly specific binding of TubR to fluorescently labeled tubC was confirmed by an EMSA that included nonspecific control DNA (Fig.…”

“…Binding of both truncated (iterons 1-3) and full-length (iterons 1-7) TubRC allowed for slow depolymerization and did not inhibit treadmilling. This effect explained the previously suggested stabilization effect of TubRC on filament formation (17,20). To check that the measured reduction in shrinkage was not caused by TubZ filament growth facilitated by bound TubRC, filaments were sequentially grown using differently labeled TubZ monomers, whereas TubR and tubC concentrations were kept constant.…”

“…We then set out to investigate the interaction of TubR with fulllength tubC DNA consisting of the complete seven iterons, because previous experiments describing the assembly of the centromeric complex used truncated tubC (iterons 4-7 only) (12,17,18). EMSAs revealed that unlabeled TubR incubated with Atto647-labeled tubC 1-7 DNA in motility buffer introduced a full shift at nanomolar concentrations.…”

“…Thus, it was proposed that the TubRC complex tracks growing ends of TubZ filaments in analogy to the centromeric complex that follows growing filament ends of the actin-like partitioning systems (5,6,19). In bulk assays, TubRC has been reported to enhance TubZ filament formation, possibly indicating a switch in dynamic behavior (17,20).…”

“…However, TubZRC alone maintains synthetic plasmids only under artificial selective pressure (7,11), and replication of pBtoxis occurs without the dnaAbox but not without TubZRC, prompting others to propose a role for the TubZRC system in plasmid replication (12). In vitro, TubZ assembles into two-and four-stranded polymers (13)(14)(15)(16), and structural studies suggested that the centromeric TubRC complex forms a ring-like structure (17), interacting with the long C-terminal TubZ extensions (18). Thus, it was proposed that the TubRC complex tracks growing ends of TubZ filaments in analogy to the centromeric complex that follows growing filament ends of the actin-like partitioning systems (5,6,19).…”