DNA gyrase is an indispensible marvelous molecular machine in manipulating the DNA topology for the prokaryotes. In the ‘two-gate’ mechanism of DNA topoisomerase, T-segment navigation from N- to DNA-gate is a critical step, but the structural basis supporting this scheme is unclear. The crystal structure of DNA gyrase B′ subfragment from Mycobacterium tuberculosis reveals an intrinsic homodimer. The two subunits, each consisting of a Tail and a Toprim domain, are tightly packed one another to form a ‘crab-like’ organization never observed previously from yeast topo II. Structural comparisons show two orientational alterations of the Tail domain, which may be dominated by a 43-residue peptide at the B′ module C-terminus. A highly conserved pentapeptide mediates large-scale intrasubunit conformational change as a hinge point. Mutational studies highlight the significant roles of a negatively charge cluster on a groove at dimer interface. On the basis of structural analysis and mutation experiments, a sluice-like model for T-segment transport is proposed.

Long noncoding RNAs (lncRNAs), which have emerged in recent years as a new and crucial layer of gene regulators, regulate various biological processes such as carcinogenesis and metastasis. HOTAIR (Hox transcript antisense intergenic RNA), a lncRNA overexpressed in most human cancers, has been shown to be an oncogenic lncRNA. Here, we explored the role of HOTAIR in HeLa cells and searched for proteins regulated by HOTAIR. To understand the mechanism of action of HOTAIR from a systems perspective, we employed a quantitative proteomic strategy to systematically identify potential targets of HOTAIR. The expression of 170 proteins was significantly dys-regulated after inhibition of HOTAIR, implying that they could be potential targets of HOTAIR. Analysis of this data at the systems level revealed major changes in proteins involved in diverse cellular components, including the cytoskeleton and the respiratory chain. Further functional studies on vimentin (VIM), a key protein involved in the cytoskeleton, revealed that HOTAIR exerts its effects on migration and invasion of HeLa cells, at least in part, through the regulation of VIM expression. Inhibition of HOTAIR leads to mitochondrial dysfunction and ultrastructural alterations, suggesting a novel role of HOTAIR in maintaining mitochondrial function in cancer cells. Our results provide novel insights into the mechanisms underlying the function of HOTAIR in cancer cells. We expect that the methods used in this study will become an integral part of functional studies of lncRNAs.

DNA gyrase plays a vital role in resolving DNA topological problems and is the target of antibiotics such as fluoroquinolones. Mycobacterium fluoroquinolone resistance protein A (MfpA) from Mycobacterium smegmatis is a newly identified DNA gyrase inhibitor that is believed to confer intrinsic resistance to fluoroquinolones. However, MfpA does not prevent drug-induced inhibition of DNA gyrase in vitro , implying the involvement of other as yet unknown factors. Here, we have identified a new factor, named Mycobacterium fluoroquinolone resistance protein B (MfpB), which is involved in the protection of DNA gyrase against drugs both in vivo and in vitro . Genetic results suggest that MfpB is necessary for MfpA protection of DNA gyrase against drugs in vivo ; an mfpB knockout mutant showed greater susceptibility to ciprofloxacin than the wild-type, whereas a strain overexpressing MfpA and MfpB showed higher loss of susceptibility. Further biochemical characterization indicated that MfpB is a small GTPase and its GTP bound form interacts directly with MfpA and influences its interaction with DNA gyrase. Mutations in MfpB that decrease its GTPase activity disrupt its protective efficacy. Our studies suggest that MfpB, a small GTPase, is required for MfpA-conferred protection of DNA gyrase.