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Regulation of RNA polymerase II termination by phosphorylation of Gdown1.

Guo J, Turek ME, Price DH - J. Biol. Chem. (2014)

Bottom Line: A highly conserved LPDKG motif found in the N-terminal domain of Gdown1 is also highly conserved in TTF2.An S270A mutation was not phosphorylated by the partially purified kinase, and an S270E mutation partially mimicked the properties of phospho-Gdown1.Gdown1 Ser-270 phosphorylation occurs predominately during mitosis, and we suggest that this would enable TTF2 to terminate all Pol II even if it is associated with Gdown1.

View Article: PubMed Central - PubMed

Affiliation: From the Department of Biochemistry, University of Iowa, Iowa City, Iowa 52242.

ABSTRACT
Gdown1 is a substoichiometric subunit of RNA polymerase II (Pol II) that has been recently demonstrated to be involved in stabilizing promoter-proximal paused Pol II. It was shown to inhibit termination of Pol II by transcription termination factor 2 (TTF2) as well as block elongation stimulation by transcription factor IIF (TFIIF). Here, using in vitro transcription assays, we identified two functional domains in Gdown1. Although both are required to maintain a tight association with Pol II, the N- and C-terminal domains are responsible for blocking TTF2 and TFIIF, respectively. A highly conserved LPDKG motif found in the N-terminal domain of Gdown1 is also highly conserved in TTF2. Deletion of this motif eliminated the TTF2 inhibitory activity of Gdown1. We identified a phosphorylated form of Gdown1 with altered mobility in SDS-PAGE that appears during mitosis. A kinase in HeLa nuclear extract that caused the shift was partially purified. In vitro, Gdown1 phosphorylated by this kinase demonstrated reduced activity in blocking both TTF2 and TFIIF because of its reduced affinity for Pol II. Mass spectrometry identified Ser-270 as the site of this phosphorylation. An S270A mutation was not phosphorylated by the partially purified kinase, and an S270E mutation partially mimicked the properties of phospho-Gdown1. Gdown1 Ser-270 phosphorylation occurs predominately during mitosis, and we suggest that this would enable TTF2 to terminate all Pol II even if it is associated with Gdown1.

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The Gdown1 CTD blocks TFIIF but not TTF2.A, silver staining of purified recombinant Gdown1 NTD(1–89). B, silver staining of purified recombinant Gdown1 CTD(197–368). C, indicated amount of Gdown1 NTD incubated with EECs before adding in TFIIF or TTF2 and chased for 7 min. D and E, indicated amounts of Gdown1 (full-length) or Gdown1 CTD(197–368) incubated with EECs and 0.1 pmol of TFIIF (D) or 0.04 pmol of TTF2 (E) as indicated before reactions were chased for 7 min. Isolated RNA was separated on a denaturing gel, and the labeled transcripts were detected by phosphorimaging.
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Figure 2: The Gdown1 CTD blocks TFIIF but not TTF2.A, silver staining of purified recombinant Gdown1 NTD(1–89). B, silver staining of purified recombinant Gdown1 CTD(197–368). C, indicated amount of Gdown1 NTD incubated with EECs before adding in TFIIF or TTF2 and chased for 7 min. D and E, indicated amounts of Gdown1 (full-length) or Gdown1 CTD(197–368) incubated with EECs and 0.1 pmol of TFIIF (D) or 0.04 pmol of TTF2 (E) as indicated before reactions were chased for 7 min. Isolated RNA was separated on a denaturing gel, and the labeled transcripts were detected by phosphorimaging.

Mentions: To assess the functions of the Gdown1 individual domains, truncation proteins containing the NTD (amino acids 1–89) and CTD (amino acids 197–364) of Gdown1 were cloned, expressed, and purified (Fig. 2, A and B). Tested in the in vitro transcription assays, a high concentration of the NTD did not block TFIIF but demonstrated a very slight inhibition of TTF2 activity (Fig. 2C), indicating that the NTD requires support from other domains of Gdown1 to have sufficient activity. The CTD has been predicted to have structural similarity to part of the RAP30 subunit of TFIIF (17), and we hypothesized that the CTD might compete with TFIIF in binding Pol II. Full-length Gdown1 and Gdown1 CTD were titrated onto EECs before adding TFIIF or TTF2 and performing a 7-min chase. Full-length Gdown1 completely blocked TFIIF at 0.02 pmol/reaction, whereas the Gdown1 CTD achieved the same blocking activity at 20 pmol (Fig. 2D). However, the CTD alone showed very slight inhibitory effect on TTF2 (Fig. 2E). Together, these results strongly suggest that the CTD of Gdown1 is primarily responsible for inhibiting TFIIF, although the full-length protein shows a much stronger block probably because of its increased ability to bind to Pol II.


Regulation of RNA polymerase II termination by phosphorylation of Gdown1.

Guo J, Turek ME, Price DH - J. Biol. Chem. (2014)

The Gdown1 CTD blocks TFIIF but not TTF2.A, silver staining of purified recombinant Gdown1 NTD(1–89). B, silver staining of purified recombinant Gdown1 CTD(197–368). C, indicated amount of Gdown1 NTD incubated with EECs before adding in TFIIF or TTF2 and chased for 7 min. D and E, indicated amounts of Gdown1 (full-length) or Gdown1 CTD(197–368) incubated with EECs and 0.1 pmol of TFIIF (D) or 0.04 pmol of TTF2 (E) as indicated before reactions were chased for 7 min. Isolated RNA was separated on a denaturing gel, and the labeled transcripts were detected by phosphorimaging.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4007455&req=5

Figure 2: The Gdown1 CTD blocks TFIIF but not TTF2.A, silver staining of purified recombinant Gdown1 NTD(1–89). B, silver staining of purified recombinant Gdown1 CTD(197–368). C, indicated amount of Gdown1 NTD incubated with EECs before adding in TFIIF or TTF2 and chased for 7 min. D and E, indicated amounts of Gdown1 (full-length) or Gdown1 CTD(197–368) incubated with EECs and 0.1 pmol of TFIIF (D) or 0.04 pmol of TTF2 (E) as indicated before reactions were chased for 7 min. Isolated RNA was separated on a denaturing gel, and the labeled transcripts were detected by phosphorimaging.
Mentions: To assess the functions of the Gdown1 individual domains, truncation proteins containing the NTD (amino acids 1–89) and CTD (amino acids 197–364) of Gdown1 were cloned, expressed, and purified (Fig. 2, A and B). Tested in the in vitro transcription assays, a high concentration of the NTD did not block TFIIF but demonstrated a very slight inhibition of TTF2 activity (Fig. 2C), indicating that the NTD requires support from other domains of Gdown1 to have sufficient activity. The CTD has been predicted to have structural similarity to part of the RAP30 subunit of TFIIF (17), and we hypothesized that the CTD might compete with TFIIF in binding Pol II. Full-length Gdown1 and Gdown1 CTD were titrated onto EECs before adding TFIIF or TTF2 and performing a 7-min chase. Full-length Gdown1 completely blocked TFIIF at 0.02 pmol/reaction, whereas the Gdown1 CTD achieved the same blocking activity at 20 pmol (Fig. 2D). However, the CTD alone showed very slight inhibitory effect on TTF2 (Fig. 2E). Together, these results strongly suggest that the CTD of Gdown1 is primarily responsible for inhibiting TFIIF, although the full-length protein shows a much stronger block probably because of its increased ability to bind to Pol II.

Bottom Line: A highly conserved LPDKG motif found in the N-terminal domain of Gdown1 is also highly conserved in TTF2.An S270A mutation was not phosphorylated by the partially purified kinase, and an S270E mutation partially mimicked the properties of phospho-Gdown1.Gdown1 Ser-270 phosphorylation occurs predominately during mitosis, and we suggest that this would enable TTF2 to terminate all Pol II even if it is associated with Gdown1.

View Article: PubMed Central - PubMed

Affiliation: From the Department of Biochemistry, University of Iowa, Iowa City, Iowa 52242.

ABSTRACT
Gdown1 is a substoichiometric subunit of RNA polymerase II (Pol II) that has been recently demonstrated to be involved in stabilizing promoter-proximal paused Pol II. It was shown to inhibit termination of Pol II by transcription termination factor 2 (TTF2) as well as block elongation stimulation by transcription factor IIF (TFIIF). Here, using in vitro transcription assays, we identified two functional domains in Gdown1. Although both are required to maintain a tight association with Pol II, the N- and C-terminal domains are responsible for blocking TTF2 and TFIIF, respectively. A highly conserved LPDKG motif found in the N-terminal domain of Gdown1 is also highly conserved in TTF2. Deletion of this motif eliminated the TTF2 inhibitory activity of Gdown1. We identified a phosphorylated form of Gdown1 with altered mobility in SDS-PAGE that appears during mitosis. A kinase in HeLa nuclear extract that caused the shift was partially purified. In vitro, Gdown1 phosphorylated by this kinase demonstrated reduced activity in blocking both TTF2 and TFIIF because of its reduced affinity for Pol II. Mass spectrometry identified Ser-270 as the site of this phosphorylation. An S270A mutation was not phosphorylated by the partially purified kinase, and an S270E mutation partially mimicked the properties of phospho-Gdown1. Gdown1 Ser-270 phosphorylation occurs predominately during mitosis, and we suggest that this would enable TTF2 to terminate all Pol II even if it is associated with Gdown1.

Show MeSH
Related in: MedlinePlus