Theme 1a : Prokarotic transcription termination
PIs : Emmanuel Margeat, Nathalie Declerck
People : Caroline Clerte (IR), Soraya Ait-Bara (post-doc)
Collaboration with M. Nollmann (CBS) and M. Boudvillain (CBM Orleans)
Our research focuses on the two mecanisms used by bacteria to terminate transcription of a gene: intrinsic termination and Rho-dependent termination. RNA transcription being a dynamic phenomenon in nature (RNA polymerase is a molecular motor, translocating along DNA while synthesizing RNA), single-molecule studies are particularly well suited for studying the molecular mechanisms involved in this central function of the cell. Most intrinsic terminators contain an inverted and repeated sequence that can base pair to itself once transcribed to create a stable terminator hairpin (7-20 bp long), that will promote RNA release upon folding. LicT and SacY from Bacillus subtilis prevent the premature arrest of transcription by binding to an antiterminator RNA hairpin that overlaps this intrinsic terminator, located in the 5’mRNA leader region of the gene to be regulated. In order to investigate the molecular determinants of this antitermination / termination balance, we have developed a fluorescence-based nucleic acids system that mimics the competition between the LicT or SacY antiterminator targets and the overlapping terminators. Using Förster Resonance Energy Transfer on single diffusing RNA hairpins, we have monitored directly their opening or closing state, and thus investigated the effects on this equilibrium of the binding of antitermination proteins or terminator-mimicking oligonucleotides. We show that the antiterminator hairpins adopt spontaneously a closed structure and that their structural dynamics is mainly governed by the length of their basal stem. The induced stability of the antiterminator hairpins determines both the affinity and specificity of the antitermination protein binding (Clerte, 2013).
The second part of this research theme focuses on the mechanisms of transcription termination by Rho, using a combination of fluorescence measurements and nanomanipulation. Rho is a hexameric helicase that uses the energy derived from ATP hydrolysis to translocate directionally along the RNA transcript, and to dissociate the transcription elongation complex. To understand the structure /function relationships giving rise to these activities, it is necessary to understand the physical basis of this translocation. Through a collaboration with Marc Boudvillain (CBM, Orleans) we showed that upon opening of a DNA / RNA hybrid model, Rho establishes specific contacts with 2’OH groups of RNA at positions separated by 7 bases (Schwartz 2009, 2012, Boudvillain 2010), and established biochemical evidence for an assymetric configuration of the hexamer upon translocation (Rahbi 2011).
Theme 1b : Transcriptional regulation in single cells
PI : Nathalie Declerck
People : Caroline Clerte (IR), Elodie Le Monnier (IE CDD)
Collaboration with the group of S. Aymerich (INRA, AgroParisTech) et O. Radulescu (DIMNP, UM2 Montpellier).
This project results from a long standing collaboration between the CBS (ND) and the team of S. Aymerich at INRA in Grignon investigating on regulatory proteins and networks in Bacillus subtilis. The aim of these researches is to unravel, from the atomic and single molecule level to the single cell and population level, the molecular bases of the transcriptional control mechanisms that underlie bacterial adaptation to environmental changes. Several transcriptional regulators have already been well characterized in vitro by combining many different approaches and technics available at the CBS or through external collaborations (e.g. with S. Sanglier in Strasbourg for mass spectrometry, G. Rivas in Madrid for analytical ultra-centrifugation, J. Stulke in Gottingen for B. subtilis engineering) (Déméné 2008, Doan 2008, Zorrilla 2008, Chaix 2010, Atmanéné 2010, Hubner 2011, Clerté 2013). Recently, we have started the characterization of these regulatory systems in live bacterial cells using state-of-the-art fluorescence microscopy methods based on multiple scanning fluctuation analysis. In the frame of the ANR grant “QuantinBaCell” (Oct 2009-June 2013), we have adapted a highly sensitive method called “2-photon scanning number and brightness” (2psN&B) analysis for counting the number of fluorescent protein molecules produced from different inducible promoters in B. subtilis reporter strains, in hundreds of individual cells under both permissive and repressive conditions (Ferguson 2011, Declerck & Royer 2013). In a case study with O. Radulescu (UM2), the stochastic activity of glycolytic and gluconeogenic gene promoters quantified in vivo by 2psN&B could be modeled and related to the repression mechanisms proposed from in vitro studies (Ferguson 2012). We also applied 2psN&B as well as “Raster Imaging Correlation Spectroscopy” (RICS) for monitoring in vivo the change in the oligomeric state and diffusion properties of fluorescent-labeled repressors upon a nutritional switch (manuscript in preparation). Finally, we have also performed in vitro studies under conditions that mimic the cellular environment. In the case of the CggR repressor, this led us to the discovery of a second effector metabolite that, in contrast to the previously identified inducer metabolite (FBP), positively modulates the DNA binding activity of this protein, probably explaining the observed discrepancy between its in vivo and in vitro behavior (manuscript in preparation).