Lionne, C. | Search bioRxiv

bioRxiv2023

wTSA-CRAFT : an open-access web server for rapid analysis of thermal shift assay experiments

The automated data processing provided by the TSA-CRAFT tool enables now to reach high throughput speed analysis of thermal shift assays. While the software is powerful and freely available, it still requires installation process and command line efforts that could be discouraging. To simplify the procedure, we decided to make it available and easy to use by implementing it with a graphical interface via a web server, enabling a cross-platform usage from any web browsers. We developed a web server embedded version of the TSA-CRAFT tool, enabling a user-friendly graphical interface for formatting and submission of the input file and visualization of the selected thermal denaturation profiles. We describe a typical case study of buffer condition optimization of the biologically relevant APH(3)-IIb bacterial protein in a 96 deep-well thermal shift analysis screening. wTSA-CRAFT is freely accessible for non-commercial usage at https://bioserv.cbs.cnrs.fr/TSA_CRAFT.

bioinformatics↗

bioRxiv2022

Structural basis of the mycobacterial stress-response RNA polymerase auto-inhibition via oligomerization

Self-assembly of macromolecules into higher-order symmetric structures is fundamental for the regulation of biological processes. Higher-order symmetric structure self-assembly by the gene expression machinery, such as bacterial DNA-dependent RNA polymerase (RNAP), has never been reported before. Here, we show that the stress-response {sigma}B factor from the human pathogen, Mycobacterium tuberculosis, induces the RNAP holoenzyme oligomerization into a supramolecular complex composed of eight RNAP units. Cryo-electron microscopy revealed a pseudo-symmetric structure of the RNAP octamer in which RNAP protomers are captured in an auto-inhibited state and display an open-clamp conformation. The structure shows that {sigma}B is sequestered by the RNAP flap and clamp domains. The transcriptional activator RbpA prevented octamer formation by promoting the initiation-competent RNAP conformation. Our results revealed that nonconserved region of {sigma} is an allosteric controller of transcription initiation and demonstrated how basal transcription factors can regulate gene expression by modulating the RNAP holoenzyme assembly and hibernation.

molecular biology↗