RNA Polymerase II Elongator Holoenzyme Is Composed of Two Discrete Subcomplexes

Abstract

Elongator is a histone acetyltransferase complex that associates with the elongating form of RNA polymerase II. We purified Elongator to virtual homogeneity via a rapid three-step procedure based largely on affinity chromatography. The purified factor, holo-Elongator, is a labile six-subunit factor composed of two discrete subcomplexes: one comprised of the previously identified Elp1, Elp2, and Elp3 proteins and another comprised of three novel polypeptides, termed Elp4, Elp5, and Elp6. Disruption of the yeast genes encoding the new Elongator proteins confers phenotypes indistinguishable from those previously described for the other elp mutants, and concomitant disruption of genes encoding proteins in either subcomplex does not confer new phenotypes. Taken together, our results indicate that holo-Elongator is a functional entity in vitro as well as in vivo. Metazoan homologues of Elp1 and Elp3 have previously been reported. We cloned the human homologue of yeast ELP4 and show that this gene is ubiquitously expressed in human tissues.The form of RNA polymerase II (RNAPII) 1 responsible for transcript elongation is fundamentally different from the form that enters a promoter to form a preinitiation complex (1, 2). During initiation, RNAPII is hypo-phosphorylated and associated with the functionally conserved Mediator complex, a multisubunit factor required for regulation of transcription (3, 4). The association of RNAPII with Mediator and the general transcription factors is severed during promoter clearance, triggered by TFIIH-mediated hyperphosphorylation of the carboxyl-terminal repeat domain (CTD) of the largest RNAPII subunit (5-7). During elongation, hyperphosphorylated yeast RNAPII is associated with the Elongator complex. Elongator binds directly to RNAPII, at least partly via the CTD, and the interaction is stabilized by CTD hyperphosphorylation (8).

Architecture of the yeast Elongator complex

“…The purified Elp123 sample (Fig 2A) was stabilized with low amounts of glutaraldehyde and eluted from a gel filtration column at a volume expected for a ~610 kDa complex. This suggested that the Elp123 sub‐complex in isolation also harbors two copies of each of the three subunits, as previously described for the full complex 20, 28.…”

“…We constructed yeast strains carrying endogenously TAP‐tagged versions of Elp1 and Elp6 and purified Elongator using previously established purification protocols 20. Consistent with sub‐stoichiometric cellular amounts of Elp456 in vivo 28, 36, 37, purifications of Elp1‐TAP resulted in an excess of Elp123 sub‐complex 20, whereas Elp6‐TAP purifications resulted in reduced quantities but yielded highly pure, complete, and stoichiometric Elongator complex. Large‐scale preparations yielded sufficient amounts of pure Elongator complex and Elp123 sub‐complex to analyze their overall architecture and shape by EM.…”

“…All subunits are highly conserved among eukaryotes 21, which has also been experimentally proven by cross species complementation analyses of genes encoding individual subunits and subdomains for yeast, insects, worms, plants, and humans 15, 22, 23, 24, 25, 26. Shortly after the initial description of the three‐component Elongator sub‐complex (Elp123) 27, an additionally associated sub‐complex containing subunits Elp4, Elp5, and Elp6 28 was identified under milder purification conditions. Crystal structures are currently available for the homo‐dimer of the Elp1 C‐terminus 29, Elp2 30, and the RecA‐like Elp456 hetero‐hexamer 20, 31.…”

RETRACTED: Identification of an RNA-dependent RNA polymerase in Drosophila involved in RNAi and transposon suppression

“…Although we do not have antibody to endogenous D-elp1, S2 cells expressing V5-tagged D-elp1 and a GFP marker selectively lost D-elp1 expression when knocked down in parallel cultures, suggesting the endogenous protein was also depleted under the treatment conditions used. D-elp1 is the largest of the three subunits in the core elongator complex, and D-elp3 has been shown to have histone acetyl transferase activity that facilitates pol II transcription through chromatinized templates in vitro (16,23,24). We wanted to know if D-elp1 depletion alone was responsible for the effects on RNA silencing or if the three subunits of the core elongator complex were equally involved, suggesting a role for the elongator complex itself.…”

Axon Transport and Neuropathy

“…100 Little is known about how Elp1 functions in disease-relevant sympathetic and sensory neurons because most studies have focused on nonneuronal cells from FD patients (eg, fibroblasts and leukocytes) or they have involved Elp1 protein modulation in cells not normally involved in disease. 101e106 Elp1 is a constituent of the heterohexameric transcriptional elongator complex 107 ; therefore, it may be involved in transcriptional regulation. 102,103,108 Interestingly, however, a significant amount of Elp1 protein is found in a vesicular pattern in the cytoplasm of both nonneuronal cells 104,109 and neurons ( Figure 1B), suggesting that it has a function apart from transcription.…”