The archaeal protein, the homolog of the third subunit of the eukaryotic Elongator complex (Elp3), is catalyses the tRNA wobble uridine modification at C5. The same reaction as the eukaryotic Elongator complex. The proposed mechanism of action by Elp3 represents an unprecedented chemistry performed on acetyl-CoA in which the methyl group of the acetly-CoA is activated by the 5'-deoxyadenosyl radical. This then adds to the uridine of tRNA.
The elongator complex is a major component of the RNA polymerase II (RNAPII) holoenzyme responsible for transcriptional elongation. It binds to both naked and nucleosomal DNA, can acetylate both core and nucleosomal histones, and is involved in chromatin remodeling. It acetylates histones H3, preferentially at 'Lys-14', and H4, preferentially at 'Lys-8'. ELP3 is required for the complex integrity and for the association of the complex with nascent RNA transcript. ELP3 is thought to act as a highly conserved histone acetyltransferase (HAT) capable of acetylating core histones in vitro, however, it is clearly a multi-domain protein. The HAT activity is thought to be present only in the C-terminal GNAT domain (histone acyltransferase domain). Recent work by Defraia et al. suggest that both the histone acetyltransferase and radical S-adenosylmethionine domains are essential for function, although the exact role of the Radical SAM domain is still unclear. Defraia et al. suggest that the radical SAM domain is important for the structural integrity of the protein complex, and in yeast (previously demonstrated). However, an alternative may be that ELP3 binds and cleave SAM, as seen in the archaea M. jannaschii. It has also been shown in previous studies that the mouse ELP3 does not require the histone acyltransferase domain for zygotic paternal genome demethylation.
Wittschieben BO, Otero G, de Bizemont T, Fellows J, Erdjument-Bromage H, Ohba R, Li Y, Allis CD, Tempst P, Svejstrup JQ
A novel histone acetyltransferase is an integral subunit of elongating RNA polymerase II holoenzyme
▸ Abstract
The elongator complex is a major component of the RNA polymerase II (RNAPII) holoenzyme responsible for transcriptional elongation in yeast. Here we identify Elp3, the 60-kilodalton subunit of elongator/RNAPII holoenzyme, as a highly conserved histone acetyltransferase (HAT) capable of acetylating core histones in vitro. In vivo, ELP3 gene deletion confers typical elp phenotypes such as slow growth adaptation, slow gene activation, and temperature sensitivity. These results suggest a role for a novel, tightly RNAPII-associated HAT in transcription of DNA packaged in chromatin.
Mol Cell
1999;4(1):123-128
| PubMed ID:
10445034
Paraskevopoulou C, Fairhurst SA, Lowe DJ, Brick P, Onesti S
The Elongator subunit Elp3 contains a Fe4S4 cluster and binds S-adenosylmethionine
▸ Abstract
The Elp3 subunit of the Elongator complex is highly conserved from archaea to humans and contains a well-characterized C-terminal histone acetyltransferase (HAT) domain. The central region of Elp3 shares significant sequence homology to the Radical SAM superfamily. Members of this large family of bacterial proteins contain a FeS cluster and use S-adenosylmethionine (SAM) to catalyse a variety of radical reactions. To biochemically characterize this domain we have expressed and purified the corresponding fragment of the Methanocaldococcus jannaschii Elp3 protein. The presence of a Fe4S4 cluster has been confirmed by UV-visible spectroscopy and electron paramagnetic resonance (EPR) spectroscopy and the Fe content determined by both a colorimetric assay and atomic absorption spectroscopy. The cysteine residues involved in cluster formation have been identified by site-directed mutagenesis. The protein binds SAM and the binding alters the EPR spectrum of the FeS cluster. Our results provide biochemical support to the hypothesis that Elp3 does indeed contain the Fe4S4 cluster which characterizes the Radical SAM superfamily and binds SAM, suggesting that Elp3, in addition to its HAT activity, has a second as yet uncharacterized catalytic function. We also present preliminary data to show that the protein cleaves SAM.
Mol Microbiol.
2006;59(3):795-806
| PubMed ID:
16420352
Defraia CT, Wang Y, Yao J, Mou Z
Elongator subunit 3 positively regulates plant immunity through its histone acetyltransferase and radical S-adenosylmethionine domains
▸ Abstract
BACKGROUND:
Pathogen infection triggers a large-scale transcriptional reprogramming in plants, and the speed of this reprogramming affects the outcome of the infection. Our understanding of this process has significantly benefited from mutants that display either delayed or accelerated defense gene induction. In our previous work we demonstrated that the Arabidopsis Elongator complex subunit 2 (AtELP2) plays an important role in both basal immunity and effector-triggered immunity (ETI), and more recently showed that AtELP2 is involved in dynamic changes in histone acetylation and DNA methylation at several defense genes. However, the function of other Elongator subunits in plant immunity has not been characterized.
RESULTS:
In the same genetic screen used to identify Atelp2, we found another Elongator mutant, Atelp3-10, which mimics Atelp2 in that it exhibits a delay in defense gene induction following salicylic acid treatment or pathogen infection. Similarly to AtELP2, AtELP3 is required for basal immunity and ETI, but not for systemic acquired resistance (SAR). Furthermore, we demonstrate that both the histone acetyltransferase and radical S-adenosylmethionine domains of AtELP3 are essential for its function in plant immunity.
CONCLUSION:
Our results indicate that the entire Elongator complex is involved in basal immunity and ETI, but not in SAR, and support that Elongator may play a role in facilitating the transcriptional induction of defense genes through alterations to their chromatin.
BMC Plant Biol
2013;13(None):102-None
| PubMed ID:
23856002
Greenwood C, Selth LA, Dirac-Svejstrup AB, Svejstrup JQ
An iron-sulfur cluster domain in Elp3 important for the structural integrity of elongator
▸ Abstract
The Elongator complex functions in diverse cellular processes, such as RNA polymerase II transcription and tRNA modification. The Elp3 subunit possesses a C-terminal histone acetyltransferase (HAT) domain and an N-terminal sequence that resembles an iron-sulfur (FeS) cluster motif. The HAT domain is well characterized, but the role of the FeS cluster is unknown, although one report proposed that it might be involved in catalyzing histone demethylation. We investigated the importance and function of the yeast Elp3 FeS cluster by a combination of genetic and biochemical means. To minimize oxidation of the Elp3 FeS cluster during purification, we also developed a novel tandem affinity tag and an accompanying isolation procedure that enables purification of tagged proteins to virtual homogeneity within a few hours of cell disruption. Our results failed to support a role for Elongator in histone demethylation. Moreover FeS cluster integrity was not required for the HAT or RNA binding activities of Elongator. However, a fully functional FeS cluster was required for Elongator integrity and for the association of the complex with its accessory factors Kti11 and Kti12. In contrast, the association of Elongator with RNA polymerase II in chromatin was unaffected by FeS cluster mutations. Together our data support the idea that the Elp3 FeS cluster is essential for normal Elongator function in vivo primarily as a structural, rather than catalytic, domain.
J Biol Chem
2009;284(11):141-149
| PubMed ID:
18986986
Okada Y, Yamagata K, Hong K, Wakayama T, Zhang Y
A role for the elongator complex in zygotic paternal genome demethylation
▸ Abstract
The life cycle of mammals begins when a sperm enters an egg. Immediately after fertilization, both the maternal and paternal genomes undergo dramatic reprogramming to prepare for the transition from germ cell to somatic cell transcription programs. One of the molecular events that takes place during this transition is the demethylation of the paternal genome. Despite extensive efforts, the factors responsible for paternal DNA demethylation have not been identified. To search for such factors, we developed a live cell imaging system that allows us to monitor the paternal DNA methylation state in zygotes. Through short-interfering-RNA-mediated knockdown in mouse zygotes, we identified Elp3 (also called KAT9), a component of the elongator complex, to be important for paternal DNA demethylation. We demonstrate that knockdown of Elp3 impairs paternal DNA demethylation as indicated by reporter binding, immunostaining and bisulphite sequencing. Similar results were also obtained when other elongator components, Elp1 and Elp4, were knocked down. Importantly, injection of messenger RNA encoding the Elp3 radical SAM domain mutant, but not the HAT domain mutant, into MII oocytes before fertilization also impaired paternal DNA demethylation, indicating that the SAM radical domain is involved in the demethylation process. Our study not only establishes a critical role for the elongator complex in zygotic paternal genome demethylation, but also indicates that the demethylation process may be mediated through a reaction that requires an intact radical SAM domain.
Nature
2010;463(7280):554-558
| PubMed ID:
20054296
Selvadurai K, Wang P, Seimetz J, Huang RH
Archaeal Elp3 catalyzes tRNA wobble uridine modification at C5 via a radical mechanism
▸ Abstract
Approximately 25% of cytoplasmic tRNAs in eukaryotic organisms have the wobble uridine (U34) modified at C5 through a process that, according to genetic studies, is carried out by the eukaryotic Elongator complex. Here we show that a single archaeal protein, the homolog of the third subunit of the eukaryotic Elongator complex (Elp3), is able to catalyze the same reaction. The mechanism of action by Elp3 described here represents unprecedented chemistry performed on acetyl-CoA.
Nat Chem Biol
2014;10(10):810-812
| PubMed ID:
25151136
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
Structural basis for tRNA modification by Elp3 from Dehalococcoides mccartyi
▸ Abstract
During translation elongation, decoding is based on the recognition of codons by corresponding tRNA anticodon triplets. Molecular mechanisms that regulate global protein synthesis via specific base modifications in tRNA anticodons are receiving increasing attention. The conserved eukaryotic Elongator complex specifically modifies uridines located in the wobble base position of tRNAs. Mutations in Elongator subunits are associated with certain neurodegenerative diseases and cancer. Here we present the crystal structure of D. mccartyi Elp3 (DmcElp3) at 2.15-Å resolution. Our results reveal an unexpected arrangement of Elp3 lysine acetyltransferase (KAT) and radical S-adenosyl methionine (SAM) domains, which share a large interface and form a composite active site and tRNA-binding pocket, with an iron-sulfur cluster located in the dimerization interface of two DmcElp3 molecules. Structure-guided mutagenesis studies of yeast Elp3 confirmed the relevance of our findings for eukaryotic Elp3s and should aid in understanding the cellular functions and pathophysiological roles of Elongator.
Nat Struct Mol Biol
2016;23(9):794-802
| PubMed ID:
27455459
