In bacteria, transcription and translation are coupled processes, in which movement of RNA polymerase (RNAP) synthesizing mRNA is coordinated with movement of the first ribosome translating mRNA. Coupling is modulated by the transcription factors NusG–which is thought to bridge RNAP and ribosome–and NusA. Here, we report cryo-EM structures of Escherichia coli transcription-translation complexes (TTCs) containing different-length mRNA spacers between RNAP and the ribosome active-center P-site. Structures of TTCs containing short spacers show a state incompatible with NusG bridging and NusA binding (TTC-A; previously termed “expressome”). Structures of TTCs containing longer spacers reveal a new state compatible with NusG bridging and NusA binding (TTC-B) and reveal how NusG bridges and NusA binds. We propose that TTC-B mediates NusG- and NusA-dependent transcription-translation coupling.

Nanocarrier administration has primarily been restricted to intermittent bolus injections with limited available options for sustained delivery in vivo. Here, we demonstrate that cylinder-to-sphere transitions of self-assembled filomicelle (FM) scaffolds can be employed for sustained delivery of monodisperse micellar nanocarriers with improved bioresorptive capacity and modularity for customization. Modular assembly of FMs from diverse block copolymer (BCP) chemistries allows in situ gelation into hydrogel scaffolds following subcutaneous injection into mice. Upon photo-oxidation or physiological oxidation, molecular payloads within FMs transfer to micellar vehicles during the morphological transition, as verified in vitro by electron microscopy and in vivo by flow cytometry. FMs composed of multiple distinct BCP fluorescent conjugates permit multimodal analysis of the scaffold’s non-inflammatory bioresorption and micellar delivery to immune cell populations for one month. These scaffolds exhibit highly efficient bioresorption wherein all components participate in retention and transport of therapeutics, presenting previously unexplored mechanisms for controlled nanocarrier delivery.

Magnetotactic bacteria biomineralize ordered chains of uniform, membrane-bound magnetite or greigite nanocrystals that exhibit nearly perfect crystal structures and species-specific morphologies. Transmission electron microscopy (TEM) is a critical technique for providing information regarding the organization of cellular and magnetite structures in these microorganisms. However, conventional TEM can only be used to image air-dried or vitrified bacteria removed from their natural environment. Here we present a correlative scanning TEM (STEM) and fluorescence microscopy technique for imaging viable cells of Magnetospirillum magneticum strain AMB-1 in liquid using an in situ fluid cell TEM holder. Fluorescently labeled cells were immobilized on microchip window surfaces and visualized in a fluid cell with STEM, followed by correlative fluorescence imaging to verify their membrane integrity. Notably, the post-STEM fluorescence imaging indicated that the bacterial cell wall membrane did not sustain radiation damage during STEM imaging at low electron dose conditions. We investigated the effects of radiation damage and sample preparation on the bacteria viability and found that approximately 50% of the bacterial membranes remained intact after an hour in the fluid cell, decreasing to ~30% after two hours. These results represent a first step toward in vivo studies of magnetite biomineralization in magnetotactic bacteria.

AbstractIn bacteria, transcription and translation are coupled processes, in which movement of RNA polymerase (RNAP) synthesizing mRNA is coordinated with movement of the first ribosome translating mRNA. Coupling is modulated by the transcription factors NusG--which is thought to bridge RNAP and ribosome--and NusA. Here, we report cryo-EM structures of Escherichia coli transcription-translation complexes (TTCs) containing different-length mRNA spacers between RNAP and the ribosome active-center P-site. Structures of TTCs containing short spacers show a state incompatible with NusG bridging and NusA binding (TTC-A; previously termed “expressome”). Structures of TTCs containing longer spacers reveal a new state compatible with NusG bridging and NusA binding (TTC-B) and reveal how NusG bridges and NusA binds. We propose that TTC-B mediates NusG- and NusA-dependent transcription-translation coupling.One Sentence SummaryCryo-EM defines states that mediate NusG- and NusA-dependent transcription-translation coupling in bacteria