The transcription elongation factor TFIIS is a component of RNA polymerase II preinitiation complexes - PubMed (original) (raw)

The transcription elongation factor TFIIS is a component of RNA polymerase II preinitiation complexes

Bong Kim et al. Proc Natl Acad Sci U S A. 2007.

Abstract

In this article, we provide direct evidence that the evolutionarily conserved transcription elongation factor TFIIS functions during preinitiation complex assembly. First, we identified TFIIS in a mass spectrometric screen of RNA polymerase II (Pol II) preinitiation complexes (PICs). Second, we show that the association of TFIIS with a promoter depends on functional PIC components including Mediator and the SAGA complex. Third, we demonstrate that TFIIS is required for efficient formation of active PICs. Using truncation mutants of TFIIS, we find that the Pol II-binding domain is the minimal domain necessary to stimulate PIC assembly. However, efficient formation of active PICs requires both the Pol II-binding domain and the poorly understood N-terminal domain. Importantly, Domain III, which is required for the elongation function of TFIIS, is dispensable during PIC assembly. The results demonstrate that TFIIS is a PIC component that is required for efficient formation and/or stability of the complex.

PubMed Disclaimer

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.

Fig. 1.

Identification of TFIIS by quantitative MS analysis of promoter DNA-purified Pol II PICs. (A) Schematic of the quantitative MS approach for the analysis of Pol II PICs. (B) Histogram of abundance ratios (+rTBP:−TBP) for 252 quantified proteins. The distributions were modeled by using a mixture model expectation–maximization algorithm. The model predicts five clusters: four clusters of TBP-stimulated proteins and one cluster of unstimulated proteins. TFIIS is a component of cluster 4. Cluster 5 (highest degree of enrichment) is not shown. (C) Quantification of isotopically labeled peptides derived from TFIIS. A SEQUEST database search (52) matched the MS/MS spectrum of an ion with mass-to-charge ratio (m/z) of 898.1 to the indicated ICAT-labeled peptide sequence corresponding to TFIIS. Relative quantification of the isotopically heavy (Lower) and normal (Upper) ICAT-labeled peptides was determined from the summed signal intensities for each peptide ion during the elution time with XPRESS software (53). The abundance ratio is the average from three independent measurements. P is the ProteinProphet score that describes the probability that the identification is correct (54).

Fig. 2.

Fig. 2.

TFIIS is a component of Pol II PICs. (A) Activator-stimulated recruitment of TFIIS to a promoter. Immobilized template assays were performed by incubating nuclear extract with the indicated templates for 40 min with or without Gal4-AH (A, 6 pmol), Gal4-VP16 (V, 4 pmol), or Gal4-GCN4 (G, 4 pmol). (B) Efficient recruitment of TFIIS and other PIC components to a promoter requires functional SAGA and Mediator complexes. Immobilized template assays were performed by incubating the indicated nuclear extracts with the HIS4 immobilized template for 40 min in the presence of Gal4-AH (1.5 pmol) (lanes 2–4). In lane 1, Dynabeads lacking DNA were incubated with Gal4-AH and nuclear extract. Five micrograms of the indicated nuclear extracts was resolved by SDS/PAGE and analyzed by Western blotting (lanes 5–7).

Fig. 3.

Fig. 3.

TFIIS stimulates formation of active PICs. PICs were assembled by incubating nuclear extracts from either a wild-type strain or a strain lacking DST1 with the indicated promoters in the presence or absence of Gal4-AH (1.5 pmol) or Gal4-VP16 (1 pmol) for 60 min. rTFIIS (3 pmol) was included where indicated. Transcription was initiated by the addition of NTPs, and reactions were stopped after 2 min. Brackets indicate correctly initiated transcripts that were detected by primer extension. The fold stimulation was calculated by comparing the transcription signal in reactions supplemented with rTFIIS to those lacking TFIIS.

Fig. 4.

Fig. 4.

TFIIS is required for efficient formation and/or stability of PICs. Western blot analysis of PIC formation in the presence and absence of TFIIS. Nuclear extract from a wild-type or a _DST1_-deletion strain was incubated with the immobilized HIS4 promoter and the activator Gal4-VP16 (1 pmol) in the presence or absence of rTFIIS (1.5 pmol, lane 5; 3 pmol, lane 6). After 60 min, the immobilized templates were washed, and protein composition was analyzed by SDS/PAGE, followed by Western blotting. Background binding to Dynabeads is shown in lanes 1 and 3. The asterisk indicates the band corresponding to rTFIIS that closely migrates with TFIIB, which was probed for in a previous experiment.

Fig. 5.

Fig. 5.

Role of the N-terminal domain and the Pol II-binding domain of TFIIS during PIC assembly. (A) Schematic representation of the TFIIS truncation mutants used in this study and summary of their activities in PIC formation and transcription assays. The amino acids defining the endpoints of each truncation mutant are indicated on the left. (B) PIC activity in the presence of TFIIS truncation mutants. PICs were assembled by incubating nuclear extracts from either a wild-type strain or a strain lacking DST1 with the HIS4 promoter template and Gal4-AH (1.5 pmol) in the absence or presence of either full-length rTFIIS (3 pmol) or the indicated rTFIIS truncation mutants (3 pmol) for 60 min. Transcription was initiated by the addition of NTPs, reactions were stopped after 2 min, and correctly initiated transcripts were detected by S1 nuclease protection. The fold stimulation was calculated by comparing the transcription signal in each reaction to that obtained in the reaction lacking TFIIS (lane 2). (C) PIC assembly in the presence of TFIIS truncation mutants. Nuclear extract from a wild-type or a _DST1_-deletion strain was incubated with the immobilized HIS4 promoter and the activator Gal4-AH (6 pmol) in the absence or presence of the indicated rTFIIS proteins (12 pmol). After 60 min, the immobilized templates were washed, and protein composition was analyzed by SDS/PAGE, followed by Western blotting. Background binding to Dynabeads is shown in lanes 1 and 2.

Similar articles

Cited by

References

    1. Hahn S. Nat Struct Mol Biol. 2004;11:394–403. - PMC - PubMed
    1. Ptashne M, Gann A. Genes and Signals. Cold Spring Harbor, NY: Cold Spring Harbor Lab Press; 2002.
    1. Kuras L, Struhl K. Nature. 1999;399:609–613. - PubMed
    1. Cosma MP. Mol Cell. 2002;10:227–236. - PubMed
    1. Zanton SJ, Pugh BF. Genes Dev. 2006;20:2250–2265. - PMC - PubMed

Publication types

MeSH terms

Substances

Grants and funding

LinkOut - more resources