Protein interaction network revealed by quantitative proteomic analysis links TFIIB to multiple aspects of the transcription cycle

Abstract

Although TFIIB is widely regarded as an initiation factor, recent reports have implicated it in multiple aspects of eukaryotic transcription. To investigate the broader role of TFIIB in transcription, we performed quantitative proteomic analysis of yeast TFIIB. We purified two different populations of TFIIB; one from soluble cell lysate, which is not engaged in transcription, and the other from the chromatin fraction which yields the transcriptionally active form of the protein. TFIIB purified from the chromatin exhibits several interactions that explain its non-canonical roles in transcription. RNAPII, TFIIF and TFIIH were the only components of the preinitiation complex with a significant presence in chromatin TFIIB. A notable feature was enrichment of all subunits of CF1 and Rat1 3' end processing-termination complexes in chromatin-TFIIB preparation. Subunits of the CPF termination complex were also detected in both chromatin and soluble derived TFIIB preparations. These results may explain the presence of TFIIB at the 3' end of genes during transcription as well as its role in promoter-termination interaction.

Keywords: Budding yeast; Proteomic analyses; RNA polymerase II; TFIIB; Transcription.

Fig. 1.

Experimental set up for purification of TFIIB from soluble and chromatin fractionations and identification of network of interacting proteins. (A) The workflow for identifying TFIIB-associated proteins in affinity purified preparations derived from soluble and chromatin fractions using tandem mass spectrometry. (B) Validation of soluble and chromatin fractions by Western blot for marker proteins. Input reflects whole cell lysate. Alpha-tubulin and histone H3 were used as marker proteins to verify authenticity of soluble and chromatin fractions. (C) Proportional Venn diagram comparing TFIIB-associated proteins in soluble and chromatin fractions. Interactors with a threshold value of 0.1 BNSAF or above only are shown here. Numbers shown here are an average of four biological replicates. Important categories of proteins present in the soluble and chromatin derived fractions is indicated.

Fig. 2.

RNAPII, TFIIF and TFIIH subunits are components of the preinitiation complex that interact with TFIIB in the chromatin fraction. (A) Schematic depiction of the preinitiation complex (PIC). UAS is the upstream activating sequence. (B) Of the twelve subunits of RNAPII, seven were consistently detected with high fidelity in the chromatin fraction. p-values calculated by the two tailed t-test indicate the level of significant enrichment of RNAPII subunits between the soluble and chromatin fractions. Error bars represent one unit of standard deviation based on four independent trials. (C) TFIIF and TFIIH subunits were the only general transcription factors consistently detected for TFIIB purified from chromatin fraction. p-values indicate significant enrichment of TFIIF subunits Tfg1 and Tfg2 in chromatin derived TFIIB.

Fig. 3.

All three termination complexes associate with TFIIB in the chromatin environment. (A) Schematic depiction of three 3′ end processing-termination complexes; CPF, CF1 and Rat1, of budding yeast with their known subunits. (B) All five subunits of the CF1 complex were significantly enriched in the TFIIB-chromatin relative to the TFIIB-soluble as evident from the p-values for individual subunits. (C) Only two of nearly fifteen subunits of CPF complex were detected in affinity purified TFIIB. p-values indicate that unlike CF1 complex subunits, CPF subunits were not enriched in chromatin derived TFIIB. (D) All three subunits of the Rat1 complex were significantly enriched in TFIIB-chromatin relative to the TFIIB-soluble preparation as evident from their respective p-values. p-values were calculated by the two tailed t-test. They indicate the level of enrichment of termination factors between the soluble and chromatin fractions. Error bars represent one unit of standard deviation based on four independent trials.

Fig. 4.

Splicing factors as well as Lsm and Arp2/3 complexes interact with TFIIB purified from chromatin. (A) Three splicing factors; Prp19, Prp43 and Sub2 were consistently detected in affinity purified TFIIB preparation. Sub2 is the only splicing factor which is significantly enriched in the chromatin fraction. (B) Subunits of the Lsm decaysome complex were consistently detected in TFIIB-chromatin preparation. p-values indicate significant enrichment of the complex in chromatin derived TFIIB. (C) Arp2/3 complex subunits exhibited strong interaction with TFIIB in the chromatin environment. (D) Coimmunoprecipitation of Arp2-HA with Rpb1 subunit of RNAPII. Western blot analysis shows the presence of Arp2-HA in both soluble and chromatin fractions, but only chromatin-linked Arp2 interacts with Rpb1. (E) Coimmunoprecipitation of Arp3-HA with Rpb1 subunit of RNAPII. Western blot analysis shows the presence of Arp3-HA in both soluble and chromatin fractions, but only chromatin-linked Arp3 interacts with Rpb1.

Fig. 5.

Model showing multiple interactions of TFIIB which allow it to function at different steps of the transcription cycle. Depicted throughout is a DNA template representative of a gene with two exons (blue), one intron (yellow), green promoter (P) region, and red terminator (T) region. In the PIC, TFIIB exhibits strong interaction with RNPII, TFIIF and TFIIH. Proteomic analysis reported here suggest that TFIIB also interacts with the Arp2/3 complex and Lsm complex during the initiation step of transcription (top). As transition from initiation to elongation proceeds, splicing occurs cotranscriptionally and TFIIB begins to interact with the splicing factors Prp19, Prp43, and Sub2 (middle). Finally, TFIIB contacts the termination factors which facilitates termination of transcription (middle). Simultaneous interaction of TFIIB with the promoter and the terminator-bound factors results in the gene assuming a looped architecture (bottom). Proximity of the terminator and promoter in the gene loop places termination factors in the vicinity of the promoter thereby conferring promoter directionality. Multiple interactions of TFIIB with initiation, splicing and termination factors allow TFIIB to perform multiple roles in the transcription cycle.