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.

Ribosomes stall when they encounter the end of mRNA without an in-frame stop codon. In bacteria, these nonstop complexes can be rescued by alternative ribosome-rescue factor A (ArfA). Here, using electron cryomicroscopy, we have determined structures of ArfA bound to the ribosome with 3′-truncated mRNA to resolutions from 3.0 to 3.4 Å. ArfA binds within the ribosomal mRNA channel and substitutes for the absent stop codon in the A site by specifically recruiting release factor 2 (RF2), initially in a compact pre-accommodated state. A similar conformation of RF2 may occur on stop codons, suggesting a general mechanism for releasefactor-mediated translational termination in which a conformational switch leads to peptide release only when the appropriate signal is present in the A site.In bacteria, 2-4% of mRNA transcripts lack an in-frame stop codon due to faulty transcription or nucleolytic cleavage (1). When translated, the inability to recruit release factors causes ribosomes to stall at the 3′ end of these nonstop transcripts. Translating ribosomes also stall at the 3′ end of intact transcripts when a stop codon is either read through or bypassed by translational frameshifting (2, 3). As the accumulation of stalled ribosomes is potentially lethal (4), bacteria have evolved various mechanisms to rescue these complexes (2, 3). Trans-translation is the primary rescue mechanism, present in nearly all sequenced bacterial species, and redirects ribosomes to resume translation on transfermessenger RNA (tmRNA). The reading frame of tmRNA encodes a degradation signal with a stop codon, which results in both recycling of the stalled ribosome and proteolysis of the aberrant polypeptide. Trans-translation is a promising target for antibiotic development (4); however, any therapeutic approach would need to circumvent the back-up mechanisms of alternative ribosome-rescue factors A (ArfA) and B (ArfB) which can allow some species of bacteria to survive in the absence of a functional trans-translation system (2, 3).ArfA in particular acts as a fail-safe for trans-translation in many bacterial species (5). In Escherichia coli, ArfA can support continued growth in the absence of trans-translation with few phenotypic consequences (5, 6). Under normal conditions, the arfA transcript is cleaved by RNase III to produce a nonstop mRNA substrate for trans-translation, resulting in the truncated ArfA protein being tagged for degradation (7-9), although a small constitutively expressed population of full-length ArfA may result from the translation of uncleaved arfA transcripts (7). When trans-translation is impaired or overwhelmed, full-length ArfA may rescue the synthesis of its truncated form. ArfA relieves stalled ribosomes through a Europe PMC Funders Author ManuscriptsEurope PMC Funders Author Manuscripts mechanism that requires peptidyl-tRNA hydrolysis by release factor 2 (RF2) but not the paralogous release factor 1 (RF1) (10,11). This is in contrast to ArfB, which shares homology with the catalytic domains of these rele...

Facial nerve (FN) injury can lead to debilitating and permanent facial paresis/paralysis (FP), where facial muscles progressively lose tone, atrophy, and ultimately reduce to scar tissue. Despite considerable efforts in the recent decades, therapies for FP still possess high failure rates and provide inadequate recovery of muscle function. In this pilot study, we used a feline model to demonstrate the potential for chronically implanted multichannel dual-cuff electrodes (MCE) to selectively stimulate injured facial nerves at low current intensities to avoid stimulus-induced neural injury. Selective facial muscle activation was achieved over six months after FN injury and MCE implantation in two domestic shorthaired cats (Felis catus). Through utilization of bipolar stimulation, specific muscles were activated at significantly lower electrical currents than was achievable with single channel stimulation. Moreover, interval increases in subthreshold current intensities using bipolar stimulation enabled a graded EMG voltage response while maintaining muscle selectivity. Histological examination of neural tissue at implant sites showed no appreciable signs of stimulation-induced nerve injury. Thus, by selectively activating facial musculature six months following initial FN injury and MCE implantation, we demonstrated the potential for our neural stimulator system to be safely and effectively applied to the chronic setting, with implications for FP treatment.

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