Activity, organization and function of the Elongator factor – Elongator
The main objectives of this project were:
1 Determination of the structural organization of the Elongator complex and of its mode of interaction with partners: The aim was to obtain the structure of individual subunit by X-ray analyses and if possible of sub-ccomplexes or of the full assembly.
2 Analysis of the biochemical activity of Elongator: We proposed to develop a biochemical assay to study Elongator activity in vitro.
3 Using biological diversity to investigate Elongator function: We proposed to use complementation between distant species using divergent systems to assay Elongator activity.
4 Characterization of molecular defects resulting from Elongator dysfunction in cells: We aimed to understand how alteration of Elongator activity affects cellular functions in yeast and eventually in mammalian cells.
- We obtained the structure of the Elp2 subunit. We also mapped interacting region linking the Elp4/5/6 subcomplex to subdomains of Elp1. The Elongator complex was also purified from yeast and, thanks to technological improvement in structural electron microscopy, we obtained envelopes of the full complex albeit at low resolution. Yeast strains with specifically tagged subunits were produced to allow the location of individual components in the full assembly. Turning to more distant organism, we identified a homologue of one subunit in bacteria, expressed it and solved its structure. A manuscript on this protein is submitted for publication.
- Focusing on Elongator partners and combining sequence similarities and information from the literature, we hypothesized that the role of the Kti11-Kti13 Elongator co-factor was to deliver electrons to the catalytic subunit of the complex. This electron transfer would be a prerequisite for the biochemical reaction. To support this conclusion, we obtained the structure of the Kti11-Kti13 dimer and established its electron transfer activity. These results have been published.
- We attempted to reconstitute Elongator activity in vitro using yeast, bacterial and archaeal proteins. We were unsuccessful so far. A competitor published the reconstitution of Elongator-like activity using an archaeal protein. Despite many efforts, we have been unable to reproduce these data. In particular, we developed anaerobic protein purification to protect the iron-sulfur cluster from oxygen-induced damage. This cumbersome procedure helped obtaining better quality protein.
- We attempted to complement defects if yeast Elongator by overexpression of an archaeal protein without success. We also tested whether archaeal and bacterial Elongator-like proteins can substitute and complement for a defect of a paralogous E. coli tRNA purification system. No complementation was observed.
The project progresses and our understanding of Elongator function improves. Technical difficulties slow down part of the project but adapted strategies to short-cut these limitations are implemented.
Publication: Structure of the Elongator cofactor complex Kti11/Kti13 provides insight into the role of Kti13 in Elongator-dependent tRNA modification. Kolaj-Robin O, McEwen AG, Cavarelli J, Séraphin B.FEBS J. 2015 Mar;282(5):819-33. doi: 10.1111/febs.13199. Epub 2015 Feb 4.
Structural basis for tRNA modification by Elp3 from Dehalococcoides mccartyi. Glatt S, Zabel R, Kolaj-Robin O, Onuma OF, Baudin F, Graziadei A, Taverniti V, Lin TY, Baymann F, Séraphin B, Breunig KD, Müller CW. Nat Struct Mol Biol. 2016 Sep;23(9):794-802.
1. Identifying and purifying protein complexes. EMBO Practical Course on Structural Characterization of Macromolecular Complexes Grenoble, June 2-7, 2014
2. Identifying and purifying protein complexes. EMBO Practical Course on Structural Characterization of Macromolecular Complexes Grenoble, May 21-27, 2016
3. Regulatory Networks of the Eukaryotic Elongator Complex. tRNA Conference 2016. Jeju, Korea, Sept 4-8, 2016.
Architecture of the yeast Elongator complex. EMBO Conference «Molecular machines: Intregrative structural and Molecular Biology« Heidelberg 20-23 November 2016
The Elongator complex is a highly conserved 6-subunit protein assembly (Elp1-6) of eukaryotes. This complex was originally identified as a factor co-purifying with polymerase II from the yeast Saccharomyces cerevisiae. The presence of a domain of the Elp3 subunit presenting similarities to histone acetyltranferases led to experiments supporting a role of Elongator in histone acetylation. However, Elongator was since implicated in many processes including DNA demethylation, tubulin acetylation, exocytosis, tRNA modification, response to DNA damage… Interest for Elongator was further stimulated by the discovery that mutations in some of its subunits lead to human diseases. Hence, mutations leading to reduced Elp1/IKBKAP expression are responsible for a rare syndrome affecting the survival of autonomic and sensory neurons, while variants of human Elp3 and Elp4 homologs impact on other diseases affecting the nervous system. Consistent with observations made on human patients, mouse mutants demonstrated that Elp1/IKBKAP inactivation is lethal while its reduced expression leads to peripheral neuropathies. Understanding the molecular function of Elongator is therefore of prime interest to expand our general knowledge and to understand the bases of these pathologies.
The wide variety of mechanisms in which Elongator was implicated makes the quest for its biochemical activity particularly challenging. Important insights were provided by the observation that overexpression of specific tRNAs suppresses the different phenotypes observed as a result of Elongator inactivation in S. cerevisiae. The generality of this finding is supported by parallel results independently obtained in fission yeast.
Building on the TAP technology, our group initiated a few years ago a structural study of the organization of Elongator in partnership with an EMBL team. This allowed recently the determination of the X-ray structure of an Elongator subcomplex containing full Elp6 and truncated Elp4 and Elp5. This structure revealed unexpectedly that Elp4-Elp5-Elp6 form a ring structure with similarities to hexameric helicases. This finding prompted us to investigate possible activities of Elongator in nucleic acid metabolism. This demonstrated that Elongator binds specifically tRNA in a manner regulated by nucleotide hydrolysis, reinforcing the link between Elongator and tRNA modifications. Recent unpublished data from our group further tighten this connection.
Intriguingly, despite years of efforts in many laboratories, several key questions related to Elongator remain unsolved. This includes defining the organization of Elongator; understanding its biochemical function, particularly its possible role in catalyzing tRNA modification; demonstrating whether phenotypic consequences of Elongator dysfunction indirectly result from altered tRNA modification or whether Elongator activity is active in several independent pathways; providing evidence for the function of Elongator contributing to human diseases. Given the general and medical interest of Elongator, we propose to investigate several of these issues. In collaboration with our collaborator at EMBL, this project builds-up on our initial structure of Elp4-Elp5-Elp6 to expand our understanding of Elongator organization. Our group will make important efforts to define the elusive biochemical function of Elongator in tRNA modification. Phylogenetic studies and synthetic biology approaches will also be implemented to get insights into the conserved function of Elongator and related proteins, as well as to attempt to define the direct function(s) of Elongator in cells and differentiate it(them) from indirect consequences of its inactivation. Molecular assays for Elongator activity will also be developed and used to assess the consequences of reduced Elongator expression in mammalian cells, aiming at providing insights into the basis of associated pathologies.
Monsieur Bertrand SERAPHIN (Institut de Génétique et de Biologie Moléculaire et Cellulaire)
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.
IGBMC Institut de Génétique et de Biologie Moléculaire et Cellulaire
Help of the ANR 220,000 euros
Beginning and duration of the scientific project: February 2014 - 42 Months