Diverse molecules, from small antibacterial drugs to large protein toxins, are exported directly across both cell membranes of gram-negative bacteria. This export is brought about by the reversible interaction of substrate-specific inner-membrane proteins with an outer-membrane protein of the TolC family, thus bypassing the intervening periplasm. Here we report the 2.1-A crystal structure of TolC from Escherichia coli, revealing a distinctive and previously unknown fold. Three TolC protomers assemble to form a continuous, solvent-accessible conduit--a 'channel-tunnel' over 140 A long that spans both the outer membrane and periplasmic space. The periplasmic or proximal end of the tunnel is sealed by sets of coiled helices. We suggest these could be untwisted by an allosteric mechanism, mediated by protein-protein interactions, to open the tunnel. The structure provides an explanation of how the cell cytosol is connected to the external environment during export, and suggests a general mechanism for the action of bacterial efflux pumps.

Two crystal structures of the glucocorticoid receptor DNA-binding domain complexed with DNA are reported. The domain has a globular fold which contains two Zn-nucleated substructures of distinct conformation and function. When it binds DNA, the domain dimerizes, placing the subunits in adjacent major grooves. In one complex, the DNA has the symmetrical consensus target sequence; in the second, the central spacing between the target's half-sites is larger by one base pair. This results in one subunit interacting specifically with the consensus target half-site and the other nonspecifically with a noncognate element. The DNA-induced dimer fixes the separation of the subunits' recognition surfaces so that the spacing between the half-sites becomes a critical feature of the target sequence's identity.

Hfq is an RNA-binding protein that is common to diverse bacterial lineages and has key roles in the control of gene expression. By facilitating the pairing of small RNAs with their target mRNAs, Hfq affects the translation and turnover rates of specific transcripts and contributes to complex post-transcriptional networks. These functions of Hfq can be attributed to its ring-like oligomeric architecture, which presents two non-equivalent binding surfaces that are capable of multiple interactions with RNA molecules. Distant homologues of Hfq occur in archaea and eukaryotes, reflecting an ancient origin for the protein family and hinting at shared functions. In this Review, we describe the salient structural and functional features of Hfq and discuss possible mechanisms by which this protein can promote RNA interactions to catalyse specific and rapid regulatory responses in vivo.Hfq was discovered in Escherichia coli nearly half a century ago (BOX 1) and was one of the first recognized representatives of an extensive RNA-binding protein family, the members of which can be found in almost every cellular organism from all three domains of life 1 . The meta-zoan homologues of Hfq include the Sm proteins, named after the autoimmune Sm antibodies that recognize them, and the closely related Sm-like (LSm) proteins, which are also found in single-celled eukaryotes and in archaea. The characteristic feature of the collective Hfq-Sm-LSm protein family is a ring-like, multimeric quaternary architecture that supports interactions with partner macromolecules. Both Hfq and the SmLSm proteins have general roles as RNA binders that contribute to post-transcriptional regulation. The Sm-LSm proteins include central components of the mRNA-splicing machinery, scaffolds for RNA-decapping assemblies, and protective chaperones of ribosomal RNAs, small nucleolar RNAs and tRNA precursors 1 .Competing interests statement The authors declare no competing financial interests. Today, Hfq is perceived primarily as the core component of a global post-transcriptional network, in which it facilitates the short and imperfect base-pairing interactions of regulatory small RNAs (sRNAs) with trans-encoded target mRNAs. Model organisms such as E. coli or Salmonella enterica can express ~100 different sRNAs. Unlike their functional equivalents in eukaryotes -the 22-nucleotide-long microRNAs -these bacterial sRNAs are heterogeneous in size and structure. The intriguing physiological functions of Hfq and sRNAs have recently been reviewed [2][3][4][5] . FURTHER INFORMATIONThere are several general mechanisms of Hfq-mediated regulation at the levels of translation or RNA stability [2][3][4][5][6][7] , and these are summarized in FIG. 1. First, Hfq can suppress protein synthesis by aiding a cognate sRNA to bind the 5′ region of its target mRNA, thus rendering this 5′ region inaccessible for translation initiation (FIG. 1a). Conversely, Hfq can boost translation by guiding an sRNA to the 5′ region of its target mRNA in order to disrupt a secondary str...

The capacity of numerous bacterial species to tolerate antibiotics and other toxic compounds arises in part from the activity of energy-dependent transporters. In Gram-negative bacteria, many of these transporters form multicomponent ‘pumps’ that span both inner and outer membranes and are driven energetically by a primary or secondary transporter component1-7. A model system for such a pump is the acridine resistance complex of Escherichia coli1. This pump assembly comprises the outer-membrane channel TolC, the secondary transporter AcrB located in the inner membrane, and the periplasmic AcrA, which bridges these two integral membrane proteins. The AcrAB-TolC efflux pump is able to vectorially transport a diverse array of compounds with little chemical similarity, and accordingly confers resistance to a broad spectrum of antibiotics. Homologous complexes are found in many Gram-negative species, including pathogens of animals and plants. Crystal structures are available for the individual pump components2-7 and these have provided insights into substrate recognition, energy coupling and the transduction of conformational changes associated with the transport process. How the subunits are organised in the pump, their stoichiometry and the details of their interactions are not known and are under debate. In this manuscript, we present the pseudoatomic structure of a complete multidrug efflux pump in complex with a modulatory protein partner8. The model defines the quaternary organization of the pump, identifies key domain interactions, and suggests a cooperative process for channel assembly and opening. These findings illuminate the basis for drug resistance in numerous pathogenic bacterial species.

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