Gillet Group Research

Abstract

The team “Ribosome, bacteria and stress” is dedicated to the study of the macromolecular interactions involved in the translation and folding of proteins. They include the study of the ribosome, of the molecular chaperones that act upon the nascent polypeptides and of some misfolded protein conformations that aggregate and trigger conformational diseases. The dynamics of these molecular machines make the analysis of their 3-D structures and interactions difficult. This is why we use a combination of biochemical, bioinformatics and electron microscopy techniques. 

For the next contract (2017-2021) the team will mainly switch to the investigation of “structures and functions of the RIBOSOME under stress conditions”.

The ribosome is the fundamental ribonucleoprotein complex that mediates protein synthesis in all living cells. At a first level we will study ribosome biogenesis in bacteria. The precursors to the small and large ribosomal subunits accumulate under severe heat stress. We will use these conditions to purify these assembly intermediates and identify their structures and composition. At a second level, we will study quality control events during trans-translation, the main system ensuring the recycling of stalled translating ribosomes and the degradation of incomplete nascent proteins when incomplete messenger RNAs (mRNAs) are translated under several stress conditions. This should lead to the presentation of a near-complete mechanistic model of trans-translation at the molecular and cellular levels. Strikingly, trans-translation is essential to the survival of many pathogenic bacteria. At a third level, we will screen molecules specifically targeting the trans-translational activity of bacterial cells. We will validate the molecular and cellular effectiveness of the molecules in vitro and in vivo on a large panel of pathogenic bacteria, including multi-resistant hospital strains which pose a major public health problem.

Ribosome biogenesis

 

Cryo-imaging of 21S particles (precursors to small 30S subunits)

The ribosome is an essential ribonucleoprotein enzyme responsible for protein synthesis, and its biogenesis is a fundamental process in all living cells. Though, the details of the fine-tuned assembly process still remain unknown. Investigations focus on elucidating the cellular processes that facilitate biogenesis of the bacterial ribosomal subunits in vivo. Towards this aim, authentic precursors, (not degraded or dead-end particles) to large 50S subunits and to small 30S subunits are overproduced in Escherichia coli. Using genetic screens, single particle analysis from cryo-EM images and proteomic data we will describe the structures and composition of the assembly intermediates. We also plan to analyse the influence of stress on ribosomal modifications and to study the impact of these modifications on both efficiency and specialisation of ribosomes for selective translation of stress mRNAs.

Structural aspects of Ribosome rescue 

Quality control in protein synthesis involves many factors to regulate or repair the ribosome during translation

We conduct the studies of ribosomal complexes ongoing trans-translation by cryo-electron microscopy: cryo-EM, imaging and single particle reconstruction. We will focus specifically on the late steps of trans-translation, Nowadays, the “resolution revolution” of cryo-electron microscopy methods allows to make it possible to get trans-translational ribosomal image reconstructions at a resolution of <4Å. Our expertise in biomolecular modeling and “in-house” computational facility will allow for the reconstruction process of the entire structure inside the electron density map.

We will also focus on the three dimensional organization of prokaryotic native polysomes under stress conditions, when the rescue systems are absent or overwhelmed. High-pressure freezing of cells over-producing polysomes and cryo-electron tomography of vitreous sections (TOVIS) will be used to identify the topology of polysomes required to maintain optimal translation efficiency and cell survival.

A new class of antibiotics inhibitting bacterial trans-trasnlation

Trans-translation as a target for future antibiotics

We conduct a medicinal chemistry program based on the structure–activity relationship (SAR) of several new compounds. To screen the molecules targeting the trans-translational activity of bacterial cells, we have developed new and specific in vitro and in vivo systems. Following this initial analysis, toxicity and effect of different molecules against bacteria harvested from clinical pathogenic isolates will be tested. In addition, synergy studies with antibiotics typically used in therapy will be conducted. 

Running this ambitious project implies combining complementary expertise. The transdisciplinarity of our team’s permanent staff as key members is a must for the success of the project, as they have a strong background and expertise in biochemistry, microbiology, cryo-EM studies of macromolecular complexes, and molecular modelling.
 

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