Flagella, the rotary propellers on the surface of bacteria, present a paradigm for how cells build and operate complex molecular nanomachines. body, hook and filament – that are assembled sequentially. A central channel runs through these substructures to the flagellum tip. The basal body houses the flagellar export machinery (A) and the rod (FliE, FlgB, FlgC, FlgF and FlgG), which extends from the inner membrane (IM) and crosses the periplasm, peptidoglycan (PG) cell wall and outer membrane (OM). The cell surface hook (FlgE) is usually a flexible universal joint that connects the external flagellum filament (flagellin, FliC) to the basal body. (A) Subunits of the rod, hook and filament link head-to-tail at the cytoplasmic membrane export machinery. Subunits are first unfolded by the export ATPase complex (red) and then dock at the FlhB export gate (orange). The N-terminal helix of the docked subunit is usually then captured by the free C-terminal helix of an exiting subunit in the flagellum channel. (B) Sequential subunits are linked head-to-tail in a chain by juxtaposed terminal helices forming parallel coiled-coils. The resulting chain Lenvatinib distributor of unfolded subunits is usually connected through the flagellum central channel to the distal tip of the flagellum. (C) The subunits in the chain transit from the gate to the tip. Subunits fold and incorporate into the flagellum tip beneath cap foldases (FlgJ for rod, FlgD for hook and FliD for filament subunit assembly). Subunit folding and/or crystallization not only provides a strong anchor at the flagellum tip, it also shortens the chain in the channel thus exerting a pulling force on the next subunit at the export gate, pulling it from the gate. The pulling force then drops rapidly as the new unfolded subunit enters the channel. This process repeats for each subunit captured into the chain. We set out a series of data that together describe a growth mechanism that PB1 is energised intrinsically. Firstly, prior to entering the flagellar central channel, free subunits dock at the cell membrane export machinery, specifically via a conserved subunit targeting motif that binds to an uncovered hydrophobic pocket around the machinery export gate component FlhB. Each docked subunit is usually then sequentially captured from the gate by linking to the free C-terminal tail of the preceding subunit, the N-terminus of which has already joined the flagellar channel (Physique 1a). The N- and C-terminal helices of these adjacent subunits are Lenvatinib distributor predicted to link by forming a parallel coiled-coil (Physique 1b), with each subunit contributing 14-32 residues. Successive linkage allows a subunit chain to form. Each newly-linked subunit is usually then pulled from the gate into the flagellar channel by the thermal motion of the unfolded subunit chain anchored at its other end at the flagellum tip. Repeated folding of subunits into the growth tip (Physique 1c) not only provides directionality to subunit transit, it also causes the chain to shorten and thus stretch, exerting an increasing pulling force on the next subunit at the gate, eventually pulling it off the gate and into the channel. The vacated export Lenvatinib distributor gate is usually then free to bind a new incoming subunit that again links to the growing chain. In this way, successive rounds of subunit linking at the cell membrane export machinery are coupled to subunit crystallization at the tip to allow continuous subunit transit and constant rate growth of the flagellum. The proposed chain mechanism imposes a strict prediction around the forces underlying the respective stages of subunit passage, in particular that subunit anchoring at the flagellum tip is usually stronger than the coiled-coil links between subunits in the chain, which in turn must be stronger than the subunit binding to the export gate. Our thermodynamic analysis confirmed this to be the case,.