Subunit interaction of the core human Elongator. (A) Schematic representation of N-terminally tagged human Elp1, Elp2, and Elp3. Numbers on the top indicate the N- and C-terminal amino acids of each construct. The promoters used to transcribe the DNA in vitro are indicated. (B) Elp1 interacts with Elp2 and with Elp3. In vitro translated N-terminally His-tagged Elp1 was incubated with in vitro translated N-terminally flag-tagged Elp2 and/or Elp3. The complex was immunoprecipitated with antibodies against the His-tag or Elp3 as described in Materials and Methods. Immunoprecipitates were separated by SDS/10% PAGE and analyzed by autoradiography.

Human core Elongator interacts with RNAPII (Pol II) in vitro. Elongator (100 ng) and RNAPII (300 ng) were incubated and then fractionated through a Superose-6 column as indicated in Materials and Methods. In parallel experiments, individual factors (top of each gel) were also fractionated on the column. The bottom of each gel denotes the fractions derived from the Elongator–RNAPII fractionation. The indicated fractions were blotted and probed with anti-Elp1, anti-Elp3, and anti-CTD as indicated. Elution positions of size markers are indicated on the top.

Purification of a hElongator. (A) Schematic representation of the steps used to purify a hElongator. (B) Fractions derived from each step of purification and containing approximately equal amounts of protein were resolved by electrophoresis on an SDS/4–15% polyacrylamide gel and stained with silver. (C Upper) Silver staining of an SDS/10% polyacrylamide gel containing aliquots of fractions derived from the Superose-6 gel filtration column. The subunits of core Elongator are indicated on the left, and the elution from the gel filtration column of protein markers is indicated on the top. (Lower) Western blot of the fractions derived from the gel filtration column analyzed by using antibodies specific for Elp1 and Elp3. (D) Human Elp3 is an HAT. (Upper) Schematic representation of human Elp3-conserved motifs D, A, and B of the GNAT superfamily (34) is indicated. The Q(R)XXGXG motif (central part of GNAT motif A) critical for acetyl-CoA recognition (35) is conserved in the human Elp3 subunit. Numbers on the top denote the amino acid sequence of hElp3. (Lower) HAT assay was performed by using the purified hElongator (20 and 100 ng of the gel filtration pool). P/CAF used as control is indicated. [³H]Acetylated core histones were analyzed by SDS/15% PAGE followed by fluorography.

Subcellular localization of human core-Elongator subunits. HeLa cells were fixed and stained with various fluorescent-conjugated antibodies or 0.01 mg/ml 4′,6-diamidino-2-phenylindole (DAPI) as indicated.

Human Elongator allows transcription from chromatin templates. (A) Immunoblot analysis of untreated HeLa NE and Elongator-depleted HeLa NE. Equal amounts (30 μg) of NE and Elongator-depleted NE were subjected to SDS/4–15% polyacrylamide gradient gel electrophoresis, transferred to nitrocellulose, and analyzed with antibodies to the proteins indicated. (B) Transcription from naked DNA and chromatin templates. Transcription was performed by using either normal NE or Elongator-depleted NE (NE[ΔELP]). Transcription reaction mixtures (40 μl) contained 80 μg of NE proteins and freshly assembled chromatin or histone-free DNA (100 ng) with (20, 40, and 80 ng) or without Elongator and acetyl-CoA (1.5 μM), as indicated. In addition, reactions were supplemented with Gal4-VP16. Reactions were incubated at 30°C for 30 min or as indicated. Products of the transcription reactions were separated by electrophoresis on a 6% sequencing gel.