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.
Highlights
Human Elongator complex from HeLa nuclear extracts decreases acetyl-CoA-dependent transcription through a chromatin template that can be restored by adding back purified Elongator complex [6]
In further support of a role in transcription, RNA immunoprecipitation revealed that Elongator is associated with the nascent RNA emanating from elongating RNAPII along the coding region of several yeast genes [8], and chromatin immunoprecipitation experiments from several laboratories demonstrated an association of Elongator with active genes in human cells (9 –11)
A Functional Iron-Sulfur Cluster in the Yeast Elp3 Protein— The Elp3 subunit of Elongator has a histone acetyltransferase (HAT) domain located at its C terminus [2, 4]
Summary
Human Elongator complex from HeLa nuclear extracts decreases acetyl-CoA-dependent transcription through a chromatin template that can be restored by adding back purified Elongator complex [6]. RNA interference-mediated depletion of the IB kinase complex-associated protein/ hElp subunit of Elongator in human cells results in transcription defects at numerous genes. These genes exhibit reduced histone H3 acetylation and decreased RNAPII density toward their 3Ј-end [10]. Genetic screening in yeast for mutations that confer resistance toward the otherwise lethal intracellular expression of the killer toxin (zymocin) ␥ subunit identified genes that were named TOT1–7 (toxin target) (16 –18) These genes either encode subunits of yeast Elongator or the Kti protein. We provide evidence that the Elp FeS cluster is functional and crucial for yeast Elongator but that it does not appear to be involved in the known catalytic role(s) of the complex. Elongator interactions with RNAPII in chromatin do not appear to require this motif
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