Elucidating the Dark Matter of Translation – EZOtrad

To characterize the orthologues of the EttA protein, we are applying the same experimental approach that allowed us to discover the function of the EttA protein (Boel et al., NSMB 2014, Chen et al., NSMB 2014). For the paralogues involved in antibiotic resistance, (macrolide) we identified an ABC-F protein of streptococcus which expressed in E. coli induced resistance. This heterologous approach allows us to use all the tools and protocols developed in E. coli to study the mechanism of action of the macrolide resistance of an ABC-F protein derived from a pathogenic bacterium.

In order to further study the translation of mRNAs, we used a large dataset of protein expression in E. coli to identify elements (structure, codons, nucleotide composition) of the mRNA that disturbs its translation (Boël et al., Nature 2016). The identification of these elements allows us to build a library of reporter genes that will serve as a toolbox for studying translation. These reporters will make possible to follow in vivo, the expression of sequences containing elements which slow down translation in order to: (i) confirm and study their effects on the translation mechanism, (ii) identify new translation factors that modulate their translation influences, (iii) purify ribosome complexes stalled on these elements to identify its composition.

For the first task of the project we identified the metabolites in the growth medium which influence the phenotype caused by the deletion of the ettA gene and thus began the characterization of the physiological response regulated by ettA. We have also characterized some of the domains and residues of EttA necessary for its function. Finally we show that other ABC-Fs are also involved in the regulation of mRNA translation.

With regard to the second task, we have identified by a global approach, the elements of the mRNA which reduce the translation efficiency. We will use them to create a reporter gene library, which, we hope, will allow us to identify a new translation factor.

This project aims to characterize the function of new translation factors at the molecular level. We propose to use a multidisciplinary approach to determine the function, but also the structure of the complex of some of these factors with the ribosome. Our recent study on the EttA protein has been a pioneer and has demonstrated the effectiveness of such approach to precisely determine the function and molecular mechanisms involved in ribosome control.

Understanding the molecular mechanisms of action of the ABC-Fs on the ribosome in E. coli will create the basis for designing the study of human ABC-F. Indeed, the use of E. coli allows large-scale screens to be set up. Furthermore, we have already demonstrated that the technical tools we have developed during our EttA study can be used to explore the functions of human ABC-F proteins. These have important implications for human health, mainly in the immune response directed against foreign DNA and RNA and the development of cancers.

The study of ABC-F proteins involved in antibiotic resistance in pathogenic bacteria will help us understand the mechanism by which the ABC-F protein protects the ribosome from the antibiotic. This information can help us in the development of new antibiotics.

The screening developed in the second part of the project is a genetic tool that can be used for other projects and should accelerate the discovery of the functions of new translation factors. In the long term, we believe we can identify factors that will improve the synthesis techniques of proteins.

Boël, G., Letso R. Neely H., Price W. N., Wong K.-H., Su, M., Luff, J., Valecha, M., Everett, J., Acton, T, Xiao, R., Montelione, G. T., Aalberts, D. P. & Hunt, J. F. (2016) Codon influence on protein expression in E. coli correlates with mRNA level. Nature 529, 358-363

The study of the ribosome and mRNA translation started in the 1960s. Since, the translation factors essential to recapitulate the translation process in vitro have been identified leading to the development of different systems of in vitro translation. Since 2000, the technical revolution of structural biology (x-ray crystallography and cryo-electronmicroscopy image reconstruction) allows us to have a precise view of the molecular mechanisms involved in protein synthesis catalyzed by the ribosome. Nevertheless, even if we can reproduce mRNA translation in vitro under specific conditions that are different from the cellular conditions, the mechanisms controlling the translation process in vivo remain largely unknown.

Recently, the function of the translation factor Ef-P, identified 35 years ago, has been characterized. Ef-P is necessary for the translation of mRNA sequence that encodes poly-proline (Ude et al., Science 2013; Doerfel et al., Science 2013). Other publications have described the structure and function of translation factors required for the regulation and the storage of the ribosome during stationary phase of growth (Boël et al., NSMB 2014; Polikanov et al., Science 2012). These studies and others demonstrate that we have not yet identified all the translation factors. Indeed, the precise functions of numerous proteins known to interact with the ribosome remain unknown or not well-defined. Being able to comprise a list of over 80 of those proteins in Escherichia coli shows the extent of the dark matter of translation. This research project aims to identify the function of some of those factors. The project is divided into two tasks. The first concerns the study of translation factors that are members of the ABC-F protein family. The second is the function elucidation of uncharacterized translation factors by genetic screens.

The first task consists of the extension and deepening of the study of the ABC-F family translation factor that my collaborators and I have recently characterized. We have shown that the protein EttA (an ABC-F member) modulates ribosome dynamic to regulate protein synthesis in energy-depleted cells. Since translational apparatus consumes more than half of the total cellular energy and it is thus not unexpected to find a new translation factor that regulate this process. We have characterized EttA function on the ribosome at the structural, biochemical and physiological levels. I propose now an in-depth study of EttA function at the cellar level. On the basis of some preliminary results, the main scientific questions that we will address are: the preference of EttA for specific mRNA ribosome complexes, and the involvement of EttA in the transcription and translation coupling. I also propose to study the molecular function of the 3 EttA paralogs present in E. coli. An ortholog of EttA responsible for antibiotic resistance in several pathogenic bacteria will be investigated as well.

The second task is to develop a screening methodology to identify the phenotype related to translation defects in a mutant collection. This collection will be composed of mutants of all proteins known to interact with the ribosome and for which the function remains unclear. In parallel, we will attempt a pull-down approach to identify new factors that interact with stalled ribosomes.

The overreach of the project is the installation of a research team in France dedicated to the investigation of the regulatory potential and function of new translation factors. Part of this work will be done in collaboration with my American colleagues who I have worked with on the EttA project.