The movement of core-lipopolysaccharide across the inner membrane of Gram-negative bacteria is catalysed by an essential ATP-binding cassette transporter, MsbA. Recent structures of MsbA and related transporters have provided insights into the molecular basis of active lipid transport; however, structural information about their pharmacological modulation remains limited. Here we report the 2.9 Å resolution structure of MsbA in complex with G907, a selective small-molecule antagonist with bactericidal activity, revealing an unprecedented mechanism of ABC transporter inhibition. G907 traps MsbA in an inward-facing, lipopolysaccharide-bound conformation by wedging into an architecturally conserved transmembrane pocket. A second allosteric mechanism of antagonism occurs through structural and functional uncoupling of the nucleotide-binding domains. This study establishes a framework for the selective modulation of ABC transporters and provides rational avenues for the design of new antibiotics and other therapeutics targeting this protein family.

Although a surprisingly large number of genes affect yeast telomeres, in most cases it is not known if their products act directly or indirectly. We describe a one-hybrid assay for telomere binding proteins and use it to establish that six proteins that affect telomere structure or function but which had not been shown previously to bind telomeres in vivo are indeed telomere binding proteins. A promoter-defective allele of HIS3 was placed adjacent to a chromosomal telomere. Candidate proteins fused to a transcriptional activation domain were tested for the ability to activate transcription of the telomere-linked HIS3 gene. Using this system, Rif1p, Rif2p, Sir2p, Sir3p, Sir4p, and Cdc13p were found to be in vivo telomere binding proteins. None of the proteins activated the same reporter gene when it was at an internal site on the chromosome. Moreover, Cdc13p did not activate the reporter gene when it was adjacent to an internal tract of telomeric sequence, indicating that Cdc13p binding was telomere limited in vivo. The amino-terminal 20% of Cdc13p was sufficient to target Cdc13p to a telomere, suggesting that its DNA binding domain was within this portion of the protein. Rap1p, Rif1p, Rif2p, Sir4p, and Cdc13p activated the telomeric reporter gene in a strain lacking Sir3p, which is essential for telomere position effect (TPE). Thus, the telomeric association of these proteins did not require any of the chromatin features necessary for TPE. The data support models in which the telomere acts as an initiation site for TPE by recruiting silencing proteins to the chromosome end.

Removal of a telomere from yeast chromosome VII in a strain having two copies of this chromosome often results in its loss. Here we show that there are three pathways that can stabilize this broken chromosome: homologous recombination, nonhomologous end joining, and de novo telomere addition. Both in a wild-type and a recombination deficient rad52 strain, most stabilization events were due to homologous recombination, whereas nonhomologous end joining was exceptionally rare. De novo telomere addition was relatively rare, stabilizing <0.1% of broken chromosomes. Telomere addition took place at a very limited number of sites on chromosome VII, most occurring close to a 35-base pair stretch of telomere-like DNA that is normally approximately 50 kb from the left telomere of chromosome VII. In the absence of the Pif1p DNA helicase, telomere addition events were much more frequent and were not concentrated near the 35-base pair tract of telomere-like DNA. We propose that internal tracts of telomere-like sequence recruit telomerase by binding its anchor site and that Pif1p inhibits telomerase by dissociating DNA primer-telomerase RNA interactions. These data also show that telomeric DNA is essential for the stable maintenance of linear chromosomes in yeast.

in vertebrates. In yeast, the sequence-speci®c DNAbinding protein Rap1p is thought to be essential for telomere function. In a tlc1h mutant, the templating region of the telomerase RNA gene is altered so that telomerase adds the vertebrate telomere sequence instead of the yeast sequence to the chromosome end. A tlc1h strain has short but stable telomeres and no growth defect. We show here that Rap1p and the Rap1p-associated Rif2p did not bind to a telomere that contains purely vertebrate repeats, while the TG 1±3 single-stranded DNA binding protein Cdc13p and the normally non-telomeric protein Tbf1p did bind this telomere. A chromosome with one entirely vertebrate-sequence telomere had a wild-type loss rate, and the telomere was maintained at a short but stable length. However, this telomere was unable to silence a telomere-adjacent URA3 gene, and the strain carrying this telomere had a severe defect in meiosis. We conclude that Rap1p localization to a C 3 TA 2 telomere is not required for its essential mitotic functions.

The outer membrane is an essential structural component of Gram-negative bacteria that is composed of lipoproteins, lipopolysaccharides, phospholipids, and integral β-barrel membrane proteins. A dedicated machinery, called the Lol system, ensures proper trafficking of lipoproteins from the inner to the outer membrane. The LolCDE ABC transporter is the inner membrane component, which is essential for bacterial viability. Here, we report a novel pyrrolopyrimidinedione compound, G0507, which was identified in a phenotypic screen for inhibitors of growth followed by selection of compounds that induced the extracytoplasmic σ stress response. Mutations in ,, and conferred resistance to G0507, suggesting LolCDE as its molecular target. Treatment of cells with G0507 resulted in accumulation of fully processed Lpp, an outer membrane lipoprotein, in the inner membrane. Using purified protein complexes, we found that G0507 binds to LolCDE and stimulates its ATPase activity. G0507 still binds to LolCDE harboring a Q258K substitution in LolC (LolC), which confers high-level resistance to G0507 but no longer stimulates ATPase activity. Our work demonstrates that G0507 has significant promise as a chemical probe to dissect lipoprotein trafficking in Gram-negative bacteria.

There is a critical need for new antibacterial strategies to counter the growing problem of antibiotic resistance. In Gram-negative bacteria, the outer membrane (OM) provides a protective barrier against antibiotics and other environmental insults. The outer leaflet of the outer membrane is primarily composed of lipopolysaccharide (LPS). Outer membrane biogenesis presents many potentially compelling drug targets as this pathway is absent in higher eukaryotes. Most proteins involved in LPS biosynthesis and transport are essential; however, few compounds have been identified that inhibit these proteins. The inner membrane ABC transporter MsbA carries out the first essential step in the trafficking of LPS to the outer membrane. We conducted a biochemical screen for inhibitors of MsbA and identified a series of quinoline compounds that kill through inhibition of its ATPase and transport activity, with no loss of activity against clinical multidrug-resistant strains. Identification of these selective inhibitors indicates that MsbA is a viable target for new antibiotics, and the compounds we identified serve as useful tools to further probe the LPS transport pathway in Gram-negative bacteria.

Early in the development of female mammals, one of the two X chromosomes is silenced in half of cells and the other X chromosome is silenced in the remaining half. The basis of this apparent randomness is not understood. We show that before X-inactivation, the two X chromosomes appear to exist in distinct states that correspond to their fates as the active and inactive X chromosomes. Xist and Tsix, noncoding RNAs that control X chromosome fates upon X-inactivation, also determine the states of the X chromosomes prior to X-inactivation. In wild-type ES cells, X chromosomes switch between states; among the progeny of a single cell, a given X chromosome exhibits each state with equal frequency. We propose a model in which the concerted switching of homologous X chromosomes between mutually exclusive future active and future inactive states provides the basis for the apparently random silencing of one X chromosome in female cells.

Besides providing experimental evidence that MntABC might be a potential therapeutic target for the development of antibiotics, our in vivo proteomics data will serve as a valuable basis for defining potential antigen combinations for multicomponent vaccines.