Volume 3, Issue 1, Pages 109-118 (January 1999) Elongator, a Multisubunit Component of a Novel RNA Polymerase II Holoenzyme for Transcriptional Elongation  Gabriel Otero, Jane Fellows, Yang Li, Therese de Bizemont, Annette M.G Dirac, Claes M Gustafsson, Hediye Erdjument-Bromage, Paul Tempst, Jesper Q Svejstrup  Molecular Cell  Volume 3, Issue 1, Pages 109-118 (January 1999) DOI: 10.1016/S1097-2765(00)80179-3

Figure 1 Separation of Initiating and Elongating Forms of RNAPII (A) Strategy for separating the different forms of RNAPII. Only RNAPII and the GTFs of a yeast whole-cell extract are depicted. (B) Western blots of fractionated whole-cell extract proteins. Soluble (sol.) and chromatin (chrom.) fractions from a strain expressing a HA-tagged version of Rpb1 (YLC2exa) were blotted and probed with antibodies. The factor probed for is indicated on the right and the antibody used on the left. Up to 70% of RNAPII in a yeast cell is typically found in ternary complexes (Svejstrup et al. 1997). (C) Hyperphosphorylated RNAPII from the chromatin fraction is engaged in elongation. Crude chromatin was digested with DNAse and RNAse, and subjected to gel filtration through TSK G4000SW. Fractions were assayed for nucleic acid content (DNA/RNA), blotted and probed for the presence of hyperphosphorylated RNAPII (Pol II0), and tested in nonspecific transcription assays (bottom panel). The reason for the low transcription activity of fraction 32 is unknown but could be due to an inhibitory factor coeluting in this fraction. Molecular Cell 1999 3, 109-118DOI: (10.1016/S1097-2765(00)80179-3)

Figure 2 Purification of the Elongating Form of RNAPII (A) Purification protocol. (B) Purified RNA polymerase II holoenzyme. Aliquots (200 μl) from Biosil SEC400 were TCA precipitated, fractionated by SDS-PAGE through a 10% gel, and stained with silver. The migration of size markers is indicated on the left. Peak fraction 16 is shown separately on the right, with the subunits of RNAPII as well as coeluting proteins (p150, p90, and p60) indicated. Proteins labeled with asterisks also coeluted with RNAPII, but either appeared to be substoichiometric, or did not consistently copurify. (C) Western blot of the fractions in (B), probed with anti-Elp1 antibodies and antibodies specific for phosphorylated CTD. Molecular Cell 1999 3, 109-118DOI: (10.1016/S1097-2765(00)80179-3)

Figure 3 Purification of Free Elongator and Reconstitution of the RNAPII–Elongator Complex (A) Purified, free elongator. Mono Q fractions (2 μl) were fractionated by SDS-PAGE through an 8% gel and stained with silver. Size marker migration is indicated on the right. Lane 3 (fraction 25) shows virtually homogenous elongator, consisting of Elp1 (p150), p90, and p60 (indicated on left and denoted by asterisks). The weaker showing of the p90 and p60 bands compared to Elp1 is typical when small amounts of elongator are silver stained. The lower panel shows the Mono Q fractions blotted and probed with anti-Elp1 antibodies. (B) Elongator and RNAPII can associate in vitro. Elongator (100 ng Mono Q fraction 25) and RNAPII (2–300 ng) were filtrated through Superose 6, either as individual factors (upper and lower panel, respectively), or after their mixing (middle panels). The indicated fractions were blotted and probed with anti-Elp1 or anti-Rpb3 antibodies as indicated. Elution of size markers are indicated at the bottom. (C) Superose 6 elution profile of native elongating RNAPII holoenzyme. RNAPII holoenzyme purified from chromatin (hydroxylapatite fraction 20 (see Figure 4B)) was filtrated through Superose 6 as described above. Fractions were blotted and probed with anti-Elp1 antibodies. Anti-II0 antibodies showed cross-reactivity to the same fractions (data not shown). Molecular Cell 1999 3, 109-118DOI: (10.1016/S1097-2765(00)80179-3)

Figure 4 CTD Hyperphosphorylation Is Required to Maintain the Integrity of Complexes Derived from Stoichiometric Amounts of Elongator and RNAPII in Chromatin (A) Stoichiometric amounts of elongator and RNAPII in chromatin. Four and one microliters of highly purified, stoichiometric elongator–RNAPII complex (Holo-Pol II), and 0.2, 0.5, and 2 μl of crude chromatin (Chromatin) were blotted and probed with antibodies as indicated. (B) Stable elongator–RNAPII association requires CTD phosphorylation. Hydroxylapatite fractions were blotted and probed with antibodies for the factors shown on the left. Molecular Cell 1999 3, 109-118DOI: (10.1016/S1097-2765(00)80179-3)

Figure 5 Growth Phenotypes of elp1Δ Yeast Cells (A) elp1Δ cells have a “slow-start” phenotype. Tetrad dissections of elp1Δ/ELP1 diploids are shown after 32 hr and 3 days of incubation, respectively. (B) Slow adaptation phenotype of elp1Δ cells. Wild-type and elp1Δ cells were grown in YPD and transferred to either YPD (glu→glu), or YPG (glu→gal) at time 0. Cell numbers plotted versus time are shown. (C) siiΔ/elp1Δ cells are hypersensitive to the drug 6-azauracil (6-AU). Cells of the indicated phenotype were grown in SD (−ura) and plated on either SD (−ura) plates (−6AU), or on SD (−ura) plates containing 50 μg/ml 6-AU (+6AU). Molecular Cell 1999 3, 109-118DOI: (10.1016/S1097-2765(00)80179-3)

Figure 6 RNAPII Transcription in elp1Δ Mutants (A) Delayed Gal1-10 activation in elp1Δ cells. Total RNA was extracted at the times indicated after galactose addition to cells grown in raffinose, and blotted and hybridized with the indicated probes. Autoradiographs are shown. (B) Delayed Pho5 activation in elp1Δ cells. Cells were grown in YPD before transfer to low-phosphate medium (Han et al. 1988). Total RNA was extracted at the times indicated, blotted, and hybridized with the indicated probes. Autoradiographs are shown. (C) Normal levels of expression of TUB1, ACT1, and RPB2 in elp1Δ cells. Cells were grown to midlogarithmic growth phase in YPD. Total RNA was extracted, blotted, and hybridized with the indicated probes. Autoradiographs and the 28S region of ethidium bromide stained RNA gels (load control) are shown. (D) NaCl-sensitive phenotype of elp1Δ cells. Cells were plated in a dilution series (cell number shown on the left) onto YPD plates containing the salt concentration indicated. (E), Delayed ENA1 activation in elp1Δ cells. Cells were grown in YPD before adding NaCl to 1 M final concentration. Total RNA was extracted at the times indicated, blotted, and hybridized with the indicated probes. Autoradiographs are shown. A cycling pattern of transcription is typical for the expression of several stress-inducible genes in yeast. Molecular Cell 1999 3, 109-118DOI: (10.1016/S1097-2765(00)80179-3)

Figure 7 A Model for the Coupling of CTD Hyperphosphorylation/Dephosphorylation to the Formation of Distinct RNAPII Complexes during the Transcription Cycle For simplicity, other factors involved in initiation, transcript elongation, and termination have been omitted. See text for details. Molecular Cell 1999 3, 109-118DOI: (10.1016/S1097-2765(00)80179-3)