Dohnalek, J. | Search bioRxiv

bioRxiv2020

Mycobacterial HelD is a nucleic acids-clearing factor for RNA polymerase

RNA synthesis is central to life, and RNA polymerase depends on accessory factors for recovery from stalled states and adaption to environmental changes. Here we investigated the mechanism by which a helicase-like factor HelD recycles RNA polymerase. We report a cryo-EM structure of an unprecedented complex between the Mycobacterium smegmatis RNA polymerase and HelD. The crescent-shaped HelD simultaneously penetrates deep into two RNA polymerase channels that are responsible for DNA binding and substrate delivery to the active site, thereby locking RNA polymerase in an inactive state. We show that HelD prevents non-specific interactions between RNA polymerase and DNA and dissociates transcription elongation complexes, but does not inhibit RNA polymerase binding to the initiation {sigma} factor. The liberated RNA polymerase can either stay dormant, sequestered by HelD, or upon HelD release, restart transcription. Our results provide insights into the architecture and regulation of the highly medically-relevant mycobacterial transcription machinery and define HelD as a clearing factor that removes undesirable nucleic acids from RNA polymerase.

molecular biology↗

bioRxiv2020

Demonstration of electron diffraction from membrane protein crystals grown in a lipidic mesophase after lamella preparation by focused ion beam milling at cryogenic temperatures

Electron crystallography of sub-micron sized 3D protein crystals has emerged recently as a valuable field of structural biology. In meso crystallization methods, utilizing lipidic mesophases, particularly lipidic cubic phases (LCPs), can produce high-quality 3D crystals of membrane proteins (MPs). A major step towards realising 3D electron crystallography of MP crystals, grown in meso, is to demonstrate electron diffraction from such crystals. The first task is to remove the viscous and sticky lipidic matrix, surrounding the crystals without damaging the crystals. Additionally, the crystals have to be thin enough to let electrons traverse them without significant multiple scattering. In the present work, we experimentally verified the concept that focused ion beam milling at cryogenic temperatures (cryo-FIB) can be used to remove excess host lipidic mesophase matrix, and then thin the crystals to a thickness suitable for electron diffraction. In this study, bacteriorhodopsin (BR) crystals grown in a lipidic mesophase of monoolein were used as a model system. LCP from a part of a 50-μm thick crystal, which was flash-frozen in liquid nitrogen, was milled away with a gallium FIB under cryogenic conditions, and a part of the crystal itself was thinned into a ∼210-nm thick lamella with the ion beam. The frozen sample was then transferred into an electron cryo-microscope (cryo-EM), and a nanovolume of ∼1400×1400×210 nm3 of the BR lamella was exposed to 200-kV electrons at a fluence of ∼0.06 e−/Å2. The resulting electron diffraction peaks were detected beyond 2.7-Å resolution (with mean signal-to-noise ratio <I/σ(I)> of >7) by a CMOS-based Ceta 16M camera. The results demonstrate, that cryo-FIB milling produces high quality lamellae from crystals grown in lipidic mesophases, and pave the way for 3D electron crystallography on crystals grown or embedded in highly viscous media.Synopsis Electron diffraction experiments on crystals of membrane proteins grown in lipidic mesophases have not been possible due to a thick layer of viscous crystallisation medium around the crystals. Here we show that focused ion beam milling at cryogenic temperatures (cryo-FIB milling) can remove the viscous layer, and demonstrate high-quality electron diffraction on a FIB-milled lamella of a bacteriorhodopsin 3D crystal.Competing Interest StatementThe authors have declared no competing interest.View Full Text

biophysics↗