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. © 2009 by The American Society for Biochemistry and Molecular Biology, Inc.