The helicase Rho is a molecular nanomotor, involved in the regulation of gene expression in the bacterium E. coli, whose mode of action remains poorly understood despite decades of research. In this project, we propose to elucidate its mecanism of action, using a combination of advanced methods of combinatorial chemistry and biophysics.
This project aims to elucidate the mechanism by which Rho acts to prevent the expression of a gene, stopping the transcription of DNA. It is a very general mechanism of gene regulation in bacteria such as E. Coli. In addition, Rho is the prime target for the antibiotic bicyclomycine.
Although Rho was discovered in the early 70s, its mechanism of action remains unclear. We use a set of technologies to elucidate these mechanisms: the nanomanipulation of nucleic acids by optical or magnetic tweezers , and combinatorial chemistry methods to determine in high troughput the chemical groups involved in the reactions.
- First observation of Rho translocation in real time on single moleulces. Demonstration of a tethered tracking mecanism.
- Development of a new method to measure the displacement of single magnetic beads
- Production ant purification of E. Coli RNAP, site specifically labeled with unnatural amino acids.
- DEvelopment of the trNAIP technique, applied to the Rho helicase
Project will continue as planned
1. Schwartz A., Rabhi M.,Margeat E. ,Boudvillain M Analysis of Helicase-RNA interactions using Nucleotide Analog Interference Mapping (NAIM), Methods in Enzymology (2012) 511, 149-169
2. Salas D, Gocheva V, Nöllmann M. Constructing a magnetic tweezers to monitor RNA translocaiton at the single-molecule level. Methods in Molecular Biology (2014).
3. Soares, E., Schwartz, A., Nollmann, M., Margeat, E. & Boudvillain, M. The RNA-mediated , asymmetric ring regulatory mechanism of the transcription termination Rho helicase decrypted by time-resolved Nucleotide Analog Interference Probing ( trNAIP ). Nucleic Acids Res. 42, 9270–84 (2014)
4. Rabhi, M., Gocheva, V, Jacquinot, F, Lee, A, Margeat, E. & Boudvillain, M. Mutagenesis-Based Evidence for an Asymmetric Configuration of the Ring-Shaped Transcription Termination Factor Rho. J. Mol. Biol. 405, 497–518 (2011)
Transcription is the process of DNA-directed RNA biosynthesis. It constitutes the first and most important step in the regulation of gene expression.(1, 2) Transcription is accomplished through the enzymatic activity of RNA polymerase (RNAP) but transcriptional regulation stems from intricate networks of interactions between components of the transcription complex (the RNAP, DNA template, and RNA transcript) and protein or metabolite co-factors. Bacterial RNAPs that share a high homology in sequence, structure, and function (2) with eukaryotic RNAPs offer excellent model systems for the analysis of transcription mechanisms. Factors that influence the activity of bacterial RNAPs, such as the transcription termination factor Rho from E. coli, can also influence the fate of RNAPs from higher organisms (3). Rho is a homo-hexameric protein which binds RNA, has RNA-dependent ATPase activity, and can unwind directionally RNA and RNA-DNA helices, properties that make Rho a unique example within the family of ring-shaped nucleic acid motors (4, 5). At present, the mechanisms used by the Rho motor to carry out its biological functions remain poorly understood. However, heightened interest for these mechanisms recently arose from the demonstration that Rho has genome-wide regulatory functions in E. coli (6, 7).
In this project, we propose to investigate the mechanism of transcription termination by Rho, using a unique combination of advanced single molecule biophysics methods and combinatorial chemogenetic analysis. First, through nanomanipulation experiments with magnetic and optical tweezers, we will determine the elementary physical parameters of the Rho motor (translocation velocity and burst size, inter-subunit coordination, coupling between ATP-hydrolysis and movement, etc…). Second, we will use a modified version of the Nucleotide Analog Interference Mapping (NAIM) approach, we termed time-resolved NAIM (trNAIM), to explore the kinetic framework of the various interference signals that we have previously uncovered (8) and that showed an intriguing 7nt periodicity in 2’-OH-dependent events activating the Rho helicase. This new approach will allow us to probe patterns of allosteric communication within the Rho ring, characterize the nature and dynamic rearrangement of functional Rho-RNA interactions, and assess how these features determine Rho function. Third, we will study the mechanism by which Rho interacts with RNAP to bring about transcription termination, using single molecule Förster Resonance Energy Transfer on immobilized transcription complexes. Finally, we will investigate how Rho behaves in more complex environments when other components of gene expression (such as transcription factor NusG) and/or specific inhibitors (Yaeo, Psu) are also present, or directly in living cells, by measuring the interaction of Rho with its partners using advanced fluorescence fluctuation techniques. In summary, we will use this information to test recent models for activation and mechanochemical transduction in the Rho ring, to revise the somewhat outdated models for Rho-dependent transcription termination and to provide a better, integrated view of Rho function and dynamics in higher-order regulatory contexts. These studies will be key in providing new insights into the mechanism of closely related multimeric helicases and of other motors of the RecA and AAA+ families of ring ATPases involved in chromosome segregation, DNA packaging, bacterial conjugation, and proteolysis.
1. G. Orphanides et al. Cell 108, 439 (2002).
2. R. H. Ebright, J Mol Biol 304, 687 (2000).
3. T. J. Santangelo et al., J Mol Biol 355, 196 (2006).
4. S. S. Patel, et al. Annu Rev Biophys Biomol Struct 69, 651 (2000).
5. J. P. Richardson, Biochim Biophys Acta 1577, 251 (2002).
6. C. J. Cardinale et al., Science 320, 935 (2008).
7. R. A. Mooney et al., Mol Cell 33, 97 (2009).
8. A. Schwartz et al., Nat Struct Mol Biol 16, 1309 (2009).
Monsieur Emmanuel Margeat (CNRS - DELEGATION REGIONALE LANGUEDOC-ROUSSILLON) – firstname.lastname@example.org
The author of this summary is the project coordinator, who is responsible for the content of this summary. The ANR declines any responsibility as for its contents.
CBS CNRS - DELEGATION REGIONALE LANGUEDOC-ROUSSILLON
CBM CNRS - DELEGATION REGIONALE CENTRE POITOU-CHARENTES
CBS CNRS - DELEGATION REGIONALE LANGUEDOC-ROUSSILLON
Help of the ANR 480,000 euros
Beginning and duration of the scientific project: - 48 Months