Chromosome Translocation Inflates Bacillus Forespores and Impacts Cellular Morphology.

The means by which the physicochemical properties of different cellular components together determine bacterial cell shape remain poorly understood. Here, we investigate a programmed cell-shape change during Bacillus subtilis sporulation, when a rod-shaped vegetative cell is transformed to an ovoid spore. Asymmetric cell division generates a bigger mother cell and a smaller, hemispherical forespore. The septum traps the forespore chromosome, which is translocated to the forespore by SpoIIIE. Simultaneously, forespore size increases as it is reshaped into an ovoid. Using genetics, timelapse microscopy, cryo-electron tomography, and mathematical modeling, we demonstrate that forespore growth relies on membrane synthesis and SpoIIIE-mediated chromosome translocation, but not on peptidoglycan or protein synthesis. Our data suggest that the hydrated nucleoid swells and inflates the forespore, displacing ribosomes to the cell periphery, stretching septal peptidoglycan, and reshaping the forespore. Our results illustrate how simple biophysical interactions between core cellular components contribute to cellular morphology.

Structural basis of human transcription-DNA repair coupling.

Transcription-coupled DNA repair removes bulky DNA lesions from the genome1,2 and protects cells against ultraviolet (UV) irradiation3. Transcription-coupled DNA repair begins when RNA polymerase II (Pol II) stalls at a DNA lesion and recruits the Cockayne syndrome protein CSB, the E3 ubiquitin ligase, CRL4CSA and UV-stimulated scaffold protein A (UVSSA)3. Here we provide five high-resolution structures of Pol II transcription complexes containing human transcription-coupled DNA repair factors and the elongation factors PAF1 complex (PAF) and SPT6. Together with biochemical and published3,4 data, the structures provide a model for transcription-repair coupling. Stalling of Pol II at a DNA lesion triggers replacement of the elongation factor DSIF by CSB, which binds to PAF and moves upstream DNA to SPT6. The resulting elongation complex, ECTCR, uses the CSA-stimulated translocase activity of CSB to pull on upstream DNA and push Pol II forward. If the lesion cannot be bypassed, CRL4CSA spans over the Pol II clamp and ubiquitylates the RPB1 residue K1268, enabling recruitment of TFIIH to UVSSA and DNA repair. Conformational changes in CRL4CSA lead to ubiquitylation of CSB and to release of transcription-coupled DNA repair factors before transcription may continue over repaired DNA.

Structure of SWI/SNF chromatin remodeller RSC bound to a nucleosome.

Chromatin-remodelling complexes of the SWI/SNF family function in the formation of nucleosome-depleted, transcriptionally active promoter regions (NDRs)1,2. In the yeast Saccharomyces cerevisiae, the essential SWI/SNF complex RSC3 contains 16 subunits, including the ATP-dependent DNA translocase Sth14,5. RSC removes nucleosomes from promoter regions6,7 and positions the specialized +1 and -1 nucleosomes that flank NDRs8,9. Here we present the cryo-electron microscopy structure of RSC in complex with a nucleosome substrate. The structure reveals that RSC forms five protein modules and suggests key features of the remodelling mechanism. The body module serves as a scaffold for the four flexible modules that we call DNA-interacting, ATPase, arm and actin-related protein (ARP) modules. The DNA-interacting module binds extra-nucleosomal DNA and is involved in the recognition of promoter DNA elements8,10,11 that influence RSC functionality12. The ATPase and arm modules sandwich the nucleosome disc with the Snf2 ATP-coupling (SnAC) domain and the finger helix, respectively. The translocase motor of the ATPase module engages with the edge of the nucleosome at superhelical location +2. The mobile ARP module may modulate translocase-nucleosome interactions to regulate RSC activity5. The RSC-nucleosome structure provides a basis for understanding NDR formation and the structure and function of human SWI/SNF complexes that are frequently mutated in cancer13.

Preparing samples from whole cells using focused-ion-beam milling for cryo-electron tomography.

Recent advances have made cryogenic (cryo) electron microscopy a key technique to achieve near-atomic-resolution structures of biochemically isolated macromolecular complexes. Cryo-electron tomography (cryo-ET) can give unprecedented insight into these complexes in the context of their natural environment. However, the application of cryo-ET is limited to samples that are thinner than most cells, thereby considerably reducing its applicability. Cryo-focused-ion-beam (cryo-FIB) milling has been used to carve (micromachining) out 100-250-nm-thin regions (called lamella) in the intact frozen cells. This procedure opens a window into the cells for high-resolution cryo-ET and structure determination of biomolecules in their native environment. Further combination with fluorescence microscopy allows users to determine cells or regions of interest for the targeted fabrication of lamellae and cryo-ET imaging. Here, we describe how to prepare lamellae using a microscope equipped with both FIB and scanning electron microscopy modalities. Such microscopes (Aquilos Cryo-FIB/Scios/Helios or CrossBeam) are routinely referred to as dual-beam microscopes, and they are equipped with a cryo-stage for all operations in cryogenic conditions. The basic principle of the described methodologies is also applicable for other types of dual-beam microscopes equipped with a cryo-stage. We also briefly describe how to integrate fluorescence microscopy data for targeted milling and critical considerations for cryo-ET data acquisition of the lamellae. Users familiar with cryo-electron microscopy who get basic training in dual-beam microscopy can complete the protocol within 2-3 d, allowing for several pause points during the procedure.

Structure of GPN-Loop GTPase Npa3 and Implications for RNA Polymerase II Assembly.

Biogenesis of the 12-subunit RNA polymerase II (Pol II) transcription complex requires so-called GPN-loop GTPases, but the function of these enzymes is unknown. Here we report the first crystal structure of a eukaryotic GPN-loop GTPase, the Saccharomyces cerevisiae enzyme Npa3 (a homolog of human GPN1, also called RPAP4, XAB1, and MBDin), and analyze its catalytic mechanism. The enzyme was trapped in a GDP-bound closed conformation and in a novel GTP analog-bound open conformation displaying a conserved hydrophobic pocket distant from the active site. We show that Npa3 has chaperone activity and interacts with hydrophobic peptide regions of Pol II subunits that form interfaces in the assembled Pol II complex. Biochemical results are consistent with a model that the hydrophobic pocket binds peptides and that this can allosterically stimulate GTPase activity and subsequent peptide release. These results suggest that GPN-loop GTPases are assembly chaperones for Pol II and other protein complexes.