Studies of nematode TFIIE function reveal a link between Ser-5 phosphorylation of RNA polymerase II and the transition from transcription initiation to elongation - PubMed (original) (raw)

Studies of nematode TFIIE function reveal a link between Ser-5 phosphorylation of RNA polymerase II and the transition from transcription initiation to elongation

S Yamamoto et al. Mol Cell Biol. 2001 Jan.

Abstract

The general transcription factor TFIIE plays important roles in transcription initiation and in the transition to elongation. However, little is known about its function during these steps. Here we demonstrate for the first time that TFIIH-mediated phosphorylation of RNA polymerase II (Pol II) is essential for the transition to elongation. This phosphorylation occurs at serine position 5 (Ser-5) of the carboxy-terminal domain (CTD) heptapeptide sequence of the largest subunit of Pol II. In a human in vitro transcription system with a supercoiled template, this process was studied using a human TFIIE (hTFIIE) homolog from Caenorhabditis elegans (ceTFIIEalpha and ceTFIIEbeta). ceTFIIEbeta could partially replace hTFIIEbeta, whereas ceTFIIEalpha could not replace hTFIIEalpha. We present the studies of TFIIE binding to general transcription factors and the effects of subunit substitution on CTD phosphorylation. As a result, ceTFIIEalpha did not bind tightly to hTFIIEbeta, and ceTFIIEbeta showed a similar profile for binding to its human counterpart and supported an intermediate level of CTD phosphorylation. Using antibodies against phosphorylated serine at either Ser-2 or Ser-5 of the CTD, we found that ceTFIIEbeta induced Ser-5 phosphorylation very little but induced Ser-2 phosphorylation normally, in contrast to wild-type hTFIIE, which induced phosphorylation at both Ser-2 and Ser-5. In transcription transition assays using a linear template, ceTFIIEbeta was markedly defective in its ability to support the transition to elongation. These observations provide evidence of TFIIE involvement in the transition and suggest that Ser-5 phosphorylation is essential for Pol II to be in the processive elongation form.

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Figures

FIG. 1

Sequence analysis of C. elegans TFIIEβ. (A) Comparison of ceTFIIEβ (ceIIEβ) with hTFIIEβ (hIIEβ). The serine-rich sequence (Ser-rich), a region similar to the small subunit of TFIIF, TFIIFβ (IIFβ), a hydrophobic region (Hydrophobic), a BR-HLH and a BR-HL are indicated (42). In the middle panel, identity (above) and similarity (below) between ceIIEβ and hIIEβ over each structural motif and characteristic sequence region are indicated as percentages. The numbers presented above and below the diagram indicate the amino acid residues that delimit each structure. (B) Sequence alignment of TFIIEβ from five different species. Amino acid sequences of TFIIEβ from human (H), X. laevis (X), D. melanogaster (D), C. elegans (C), and yeast S. cerevisiae (Y) were aligned. Completely identical residues are shaded in black, and residues identical in four species are shaded in gray. Conserved similar residues are shown in bold type. Identical and similar amino acids were assigned as described previously (36). Putative structural motifs and characteristic sequences are shown as described for Fig. 1A and boxed (boxes a to f), except that a ς3 homology region (box d, ς3 homology) is additionally indicated. A hyphen indicates a gap.

FIG. 2

Sequence analysis of C. elegans TFIIEα. (A) Comparison of ceTFIIEα (ceIIEα) with hTFIIEα (hIIEα). The region with ς subdomain homology and a leucine repeat (ς, LR), a zinc finger motif (ZF), a helix-turn-helix motif (HTH), a hydrophobic region (Hydrophobic), two regions rich in alanine and glycine residues (Ala, Gly-rich), and the first and second acidic regions (2nd acidic) are indicated (38). Identity (above) and similarity (below) between ceIIEα and hIIEα over each structural motif and characteristic sequence region are indicated as shown in Fig. 1A. (B) Sequence alignment of TFIIEα from five different species. Amino acid sequences of TFIIEα from four species were aligned as shown in Fig. 1B. Identical and similar amino acids were assigned as described previously (37). Putative structural motifs and characteristic sequences are shown as described for panel A and boxed (boxes a to j), except that two overlapping ς subdomain homology regions (box a, ς 2.1–2.2 homology, and box b, ς 4.1–4.2 homology) and two alanine, glycine-rich regions (box g, 1st alanine, glycine-rich, and box h, 2nd alanine, glycine-rich) are additionally indicated. A hyphen indicates a gap.

FIG. 3

Identification of natural C. elegans TFIIE. (A) Coimmunoprecipitation of natural ceTFIIEα with ceTFIIEβ. To demonstrate association of natural ceTFIIEα with ceTFIIEβ, 80 μg of C. elegans embryonic nuclear extract was incubated with anti-ceTFIIEβ antibody–protein G-Sepharose, and natural ceTFIIEβ was precipitated. After SDS-PAGE on a 10% polyacrylamide gel, coprecipitated ceTFIIEα was detected with anti-ceTFIIEα rabbit antibody after Western blotting. Lane 1, 5% input of embryonic nuclear extract (4 μg); lane 2, recombinant ceTFIIEα (20 ng); lane 3, nuclear extract treated with preimmune serum (80 μg); lane 4, nuclear extract treated with anti-ceTFIIEβ serum (80 μg). An arrow indicates the position of ceTFIIEα (ceIIEα). (B) Coimmunoprecipitation of natural ceTFIIEβ with ceTFIIEα. The same strategy as detailed for panel A was employed to study the association of natural ceTFIIEβ with ceTFIIEα. Eighty micrograms of nuclear extract was incubated with anti-ceTFIIEα antibody–protein G-Sepharose, and natural ceTFIIEα was precipitated. Coprecipitated ceTFIIEβ was detected by anti-ceTFIIEβ rabbit antibody after Western blotting. Lane 1, 25% input of embryonic nuclear extract (20 μg); lane 2, nuclear extract treated with preimmune serum (80 μg); lanes 3 and 4, increasing amounts of nuclear extract treated with anti-ceTFIIEβ serum (40 and 80 μg, respectively); lane 5, recombinant ceTFIIEβ (200 ng). An arrow indicates the position of ceTFIIEβ (ceIIEβ). (C) Transcription complementation assay of ceTFIIE. ceTFIIE was depleted from nuclear extracts by treatment with anti-ceTFIIEβ antibody–protein G-Sepharose. In the same way, mock-depleted nuclear extracts were prepared by treatment with preimmune IgG–protein G-Sepharose. Complementation of natural ceTFIIE was studied by adding increasing amounts of purified recombinant ceTFIIE and carrying out primer extension reactions. Lane 1, ceTFIIE-depleted nuclear extracts (42 μg); lane 2, mock-depleted nuclear extracts (42 μg); lanes 3 to 5, ceTFIIE-depleted nuclear extracts (42 μg) with increasing amounts of recombinant ceTFIIE (5, 10, and 20 ng, respectively); lane 6, C. elegans nuclear extracts (42 μg). Arrows indicate the positions of the reverse transcript (92 nt) and the primer.

FIG. 4

Characterization of recombinant C. elegans TFIIE. (A) SDS-PAGE analysis of four different recombinant TFIIE proteins, with subunits from either human or C. elegans. Recombinant TFIIE subunits were coexpressed in four different combinations from the coexpression plasmids described in Materials and Methods, purified, and analyzed by SDS–10% PAGE (lanes 1 to 4). The sizes of molecular mass markers are indicated on the left (in kilodaltons). The approximate positions of TFIIEα (IIEα) and TFIIEβ (IIEβ) are indicated by arrows on the right. Asterisks indicate degradation products derived from ceTFIIEα and hTFIIEβ in lane 2. (B) Basal transcription activities of chimeric TFIIE. In vitro transcription assays were carried out with increasing amounts of chimeric TFIIE proteins. Lane 1, no TFIIE (−); lanes 2 to 4, 10, 20, and 40 ng of TFIIE made up of 6H-ceTFIIEα and nontagged ceTFIIEβ (ceEαceEβ); lanes 5 to 7, 10, 20, and 40 ng of TFIIE made up of 6H-ceTFIIEα and nontagged hTFIIEβ (ceEαhEβ); lane 8, 12 ng of TFIIE made up of 6H-hTFIIEα and nontagged ceTFIIEβ (hEαceEβ); lane 9, 12 ng of TFIIE made up of 6H-hTFIIEα and nontagged hTFIIEβ (hEαhEβ); lanes 10 and 11, 3 ng of 6H-ceTFIIEβ (ceEβ) and 6H-hTFIIEβ (hEβ), respectively. The arrow indicates the position of the specific transcript (390 nt).

FIG. 5

Binding assays using C. elegans TFIIEβ. (A) Binding of hTFIIEβ to the various general transcription factors. All of the human general transcription factors (400 ng each) except for hTFIIEβ, the TBP activation factors of TFIID, and the TFIIH subunits were fused to GST and expressed in bacteria. GST-pull down assays were carried out with 200 ng of 6H-hTFIIEβ. After SDS-PAGE on a 10% polyacrylamide gel and Western blotting, bound 6H-hTFIIEβ was detected with anti-hTFIIEβ antibody. Lane 1, control bacterial lysate (no GST protein) (−); lane 2, GST alone (no fusion protein) (G); lane 3, GST-TFIIB (B); lane 4, GST-hTFIIEα (Eα); lane 5, GST-TFIIFα (Fα); lane 6, GST-TFIIFβ (Fβ); lane 7, GST-TBP (T); lane 8, GST-TFIIAα (Aα); lane 9, GST-TFIIAβ (Aβ); lane 10, GST-TFIIAγ (Aγ). An arrow indicates the position of 6H-hTFIIEβ (hIIEβ). (B) Binding of ceTFIIEβ to the various general transcription factors. GST-pull down assays were carried out as described for panel A, except that HA-ceTFIIEβ was used instead of 6H-hTFIIEβ. After Western blotting, bound HA-ceTFIIEβ was detected with anti-HA monoclonal antibody (12CA5). An arrow indicates the position of HA-ceTFIIEβ (ceIIEβ). (C) Binding of hTFIIEβ to the TFIIH subunits. The assay was done as described for panel A. Four hundred nanograms of each GST-fused TFIIH subunit, with GST alone (lane 1) as a control, were used to examine binding to hTFIIEβ. An arrow indicates the position of 6H-hTFIIEβ (hIIEβ). (D) Binding of ceTFIIEβ to the TFIIH subunits. GST-pull down assays were carried out as described for panel C, except that HA-ceTFIIEβ was used instead of 6H-hTFIIEβ. An arrow indicates the position of HA-ceTFIIEβ (ceIIEβ). Molecular mass markers are shown to the left.

FIG. 6

Binding assays using C. elegans TFIIEα. (A) Binding of hTFIIEα to the various general transcription factors. All of the human general transcription factors (400 ng each) except for hTFIIEα, the TBP activation factors of TFIID, and the TFIIH subunits were fused to GST and expressed in bacteria. GST-pull down assays were carried out as described for Fig. 5A using 200 ng of 6H-hTFIIEα. After Western blotting, bound hTFIIEα was detected with anti-hTFIIEα antibody. Lane 1, GST alone (G); lane 2, GST-TFIIB (B); lane 3, GST-hTFIIEβ (Eβ); lane 4, GST-TFIIFα (Fα); lane 5, GST-TFIIFβ (Fβ); lane 6, GST-TBP (T); lane 7, GST-TFIIAα (Aα); lane 8, GST-TFIIAβ (Aβ); lane 9, GST-TFIIAγ (Aγ). Arrows indicate the position of 6H-hTFIIEα (hIIEα). (B) Binding of ceTFIIEα to the various general transcription factors. GST-pull down assays were carried out as described for panel A using 200 ng of HA-ceTFIIEα. After Western blotting, bound HA-ceTFIIEα was detected with anti-HA monoclonal antibody (12CA5). An arrow indicates the position of HA-ceTFIIEα (ceIIEα). (C) Binding of hTFIIEα to the TFIIH subunits. Assays were carried out as described for panel A. Four hundred nanograms of each GST-fused TFIIH subunit, with GST alone (lane 1) as a control, were used to examine binding to hTFIIEα. An arrow indicates the position of 6H-hTFIIEα (hIIEα). (D) Binding of ceTFIIEα to the TFIIH subunits. Assays were carried out as described for panel C, except that HA-ceTFIIEα was used instead of 6H-hTFIIEα. An arrow indicates the position of HA-ceTFIIEα (ceIIEα). Molecular mass markers are indicated to the left.

FIG. 7

Effects of ceTFIIE subunits on CTD phosphorylation by TFIIH during preinitiation complex formation. Kinase assays (25 μl) were carried out as described in Materials and Methods. TFIIE proteins were prepared and purified as described for Fig. 4A and in Materials and Methods. (A) CTD phosphorylation of intact Pol II during PIC formation. Lane 1, kinase reaction without TFIIE (−); lanes 2 and 3, ceTFIIE (ceIIEαceIIEβ); lanes 4 and 5, chimeric TFIIE made up of ceTFIIEα and hTFIIEβ (ceIIEαhIIEβ); lanes 6 and 7, chimeric TFIIE made up of hTFIIEα and ceTFIIEβ (hIIEαceIIEβ); lanes 8 and 9, hTFIIE (hIIEαhIIEβ). For lanes 2, 4, 6, and 8, 15 ng of each TFIIE was added. For lanes 3, 5, 7, and 9, 30 ng of each TFIIE was added. Phosphorylated proteins were analyzed on a 5.5% acrylamide-SDS gel and detected by autoradiography. Arrows indicate the positions of the phosphorylated (IIo) and unphosphorylated (IIa) forms of the largest subunit of Pol II. (B) Detection of the largest subunit of Pol II after treatment with kinases. The kinase reaction was carried out essentially as described for panel A, except that nonisotopic ATP was used instead of [γ-32P]ATP and a monoclonal antibody (8WG16) against the CTD heptapeptide was used to detect the largest subunit of Pol II. (C) Detection of phosphorylated Ser-2 in the CTD of the largest subunit of Pol II. Reactions were carried out as described for panel B, and phospho-Ser-2 in the CTD was detected using the monoclonal antibody H5. (D) Detection of phosphorylated Ser-5 in the CTD of the largest subunit of Pol II. Reactions were carried out as described for panel B, and phospho-Ser-5 in the CTD was detected using the monoclonal antibody H14.

FIG. 8

Involvement of TFIIE in the transcription transition step. (A) Schematic representation of the transcription transition assay. Pol II and the general transcription factors were preincubated on either linear or supercoiled AdML template pML(C2AT)100. Transcription was initiated by the addition of nucleotides. Both templates give a 107-nt transcript. (B) Effects of ceTFIIE subunits on the transcription transition. Transcription was carried out for 20 min (odd-numbered lanes) or 45 min (even-numbered lanes) in the presence of the various TFIIE proteins listed on the top or in the absence of TFIIE. To the right of the panel, the sizes of markers are shown in nucleotides. Lane 1, reaction without TFIIE (−E); lanes 2 and 3, ceTFIIE (ceEαceEβ); lanes 4 and 5, chimeric TFIIE made up of ceTFIIEα and hTFIIEβ (ceEαhEβ); lanes 6 and 7, chimeric TFIIE made up of hTFIIEα and ceTFIIEβ (hEαceEβ); lanes 8 and 9, hTFIIE (hEαhEβ). Lanes 1 to 12, transcription on a linear template; lanes 13 to 18, transcription on a supercoiled template. Transcripts longer than 35 nt were considered to be elongating transcripts, and their amounts were measured by a Fuji-BAS2500 phosphoimager. Transcription by wild-type TFIIE on the two different templates was defined as 100% (lanes 10 and 18). Relative transcription activities (%) are presented in the bottom panel. (C) Effects of ceTFIIE subunits on transcription initiation. The PIC was preformed as described for panel A on the _Afl_III-_Sca_I fragment of pMLH1 (containing the AdML promoter sequence from −111 to +47) (14). Transcription initiation was then carried out for 45 min at 28°C by addition of [α-32P]CTP in the presence of the four different TFIIE proteins or in the absence of TFIIE. The position of the dinucleotide transcript (adenylyl cytidine [ApC]) is indicated by an arrow. Lane 1, reaction without TFIIE (−E); lanes 2, 4, 6, and 8, 8 ng of each TFIIE protein was added; lanes 3, 5, 7, and 9, 24 ng of each TFIIE protein was added. Abbreviations for TFIIE proteins were the same as those used in panel B.

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Studies of nematode TFIIE function reveal a link between Ser-5 phosphorylation of RNA polymerase II and the transition from transcription initiation to elongation - PubMed (original) (raw)