FtsK is a DNA translocase that coordinates chromosome segregation and cell division in bacteria. In addition to its role as activator of XerCD site-specific recombination, FtsK can translocate double-stranded DNA (dsDNA) rapidly and directionally and reverse direction. We present crystal structures of the FtsK motor domain monomer, showing that it has a RecA-like core, the FtsK hexamer, and also showing that it is a ring with a large central annulus and a dodecamer consisting of two hexamers, head to head. Electron microscopy (EM) demonstrates the DNA-dependent existence of hexamers in solution and shows that duplex DNA passes through the middle of each ring. Comparison of FtsK monomer structures from two different crystal forms highlights a conformational change that we propose is the structural basis for a rotary inchworm mechanism of DNA translocation.
Accurate DNA partition at cell division is vital to all living organisms. In bacteria, this process can involve partition loci, which are found on both chromosomes and plasmids. The initial step in Escherichia coli plasmid R1 partition involves the formation of a partition complex between the DNA-binding protein ParR and its cognate centromere site parC on the DNA. The partition complex is recognized by a second partition protein, the actin-like ATPase ParM, which forms filaments required for the active bidirectional movement of DNA replicates. Here, we present the 2.8 Å crystal structure of ParR from E. coli plasmid pB171. ParR forms a tight dimer resembling a large family of dimeric ribbon-helix-helix (RHH) 2 site-specific DNA-binding proteins. Crystallographic and electron microscopic data further indicate that ParR dimers assemble into a helix structure with DNA-binding sites facing outward. Genetic and biochemical experiments support a structural arrangement in which the centromere-like parC DNA is wrapped around a ParR protein scaffold. This structure holds implications for how ParM polymerization drives active DNA transport during plasmid partition.
Localization of proteins in cells or complexes using electron microscopy has mainly relied upon the use of heavy metal clusters, which can be difficult to direct to sites of interest. For this reason, we would like to develop a clonable tag analogous to the clonable fluorescent tags common to light microscopy. Instead of fluorescing, such a tag would initiate formation of a heavy metal cluster. To test the feasibility of such a tag, we exploited the metal-binding protein, metallothionein (MT). We created a chimeric protein by fusing one or two copies of the MT gene to the gene for maltose binding protein. These chimeric proteins bound many gold atoms, with a conservative value of 16 gold atoms per copy of metallothionein. Visualization of gold-labeled fusion proteins by scanning electron microscopy required one copy of metallothionein while transmission electron microscopy required two copies. Images of frozen-hydrated samples of simple complexes made with anti-MBP antibodies hint at the usefulness of this method.
Electron cryotomography provides nanometer resolution structural detail of thin biological specimens in a near-native state. Currently, its application is limited by the lack of a specific label for the identification of molecules. Our aim was to develop such a label, analogous to GFP used in fluorescence microscopy. Here, we demonstrate the use of Escherichia coli ferritin FtnA protein as a clonable label for electron cryotomography. Overproduced ferritin is visible in E. coli cells using cryotomography and fusing this label to a short membrane targeting sequence correctly directs the ferritin fusion to the membrane. Using two proteins with known subcellular localization patterns with this ferritin tag, also including GFP, we obtained essentially the same labeling patterns using electron cryotomography as compared with fluorescence light microscopy. Hence, the ferritin label localizes efficiently and faithfully and it will be a valuable tool for the unambiguous identification of molecules in cellular electron cryotomograms.
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