Initiator-Directed Transcription: Fission Yeast Nmtl Initiator Directs Preinitiation Complex Formation and Transcriptional Initiation

Abstract

The initiator element is a core promoter element encompassing the transcription start site, which is found in yeast, Drosophila, and human promoters. This element is observed in TATA-less promoters. Several studies have defined transcription factor requirements and additional cofactors that are needed for transcription initiation of initiator-containing promoters. However, those studies have been performed with additional core promoters in addition to the initiator. In this work, we have defined the pathway of preinitiation complex formation on the fission yeast nmt1 gene promoter, which contains a functional initiator with striking similarity to the initiator of the human dihydrofolate reductase (hDHFR) gene and to the factor requirement for transcription initiation of the nmt1 gene promoter. The results show that the nmt1 gene promoter possesses an initiator encompassing the transcription start site, and several conserved base positions are required for initiator function. A preinitiation complex formation on the nmt1 initiator can be started by TBP/TFIIA or TBP/TFIIB, but not TBP alone, and afterwards follows the same pathway as preinitiation complex formation on TATA-containing promoters. Transcription initiation is dependent on the general transcription factors TBP, TFIIB, TFIIE, TFIIF, TFIIH, RNA polymerase II, Mediator, and a cofactor identified as transcription cofactor for initiator function (TCIF), which is a high-molecular-weight protein complex of around 500 kDa. However, the TAF subunits of TFIID were not required for the nmt1 initiator transcription, as far as we tested. We also demonstrate that other initiators of the nmt1/hDHFR family can be transcribed in fission yeast whole-cell extracts.

Keywords: RNA polymerase II; Schizosaccharomyces pombe; general transcription factors (GTFs); initiator; transcription.

Figure 1

Fission yeast TATA-less promoters contain a highly conserved sequence around the TSS. (A) Several fission yeast TATA-less promoters (obtained from the EPD) were aligned with the nmt1 Inr and the human DHFR Inr. The conserved sequence (TTCA + 1ACTT), that represents the Inr, is highlighted in red. The alignment was performed with the ClustalW program (https://www.genome.jp/tools-bin/clustalw, accessed on 23 April 2021). (B) Distribution of the TTCAACTT sequence in S. pombe (obtained from the EPD). It can be observed that the exact match to this sequence is contained in approximately 4% of the fission yeast promoters described in S. pombe in the EPD (https://epd.epfl.ch, accessed on 12 June 2021).

Figure 2

The nmt1 Inr is transcribed by RNAPII in WCE. (A) The nmt1 gene promoter containing a TATA box and an Inr is transcribed in WCE, and the transcription is inhibited by α-Amanitin. Concentrations of 0.5, 1.0, 2.0, 4.0, and 8.0 µg/mL were used to inhibit the transcription of the TATA box Inr-containing nmt1 promoter (wt TATA Inr). It can be observed that concentrations over 4.0 µg/mL of α-Amanitin completely inhibited the transcription of the wild-type TATA box Inr-containing promoter. On the other hand, concentrations of 2.0, 4.0, and 8.0 µg/mL of α-Amanitin completely inhibited transcription of the nmt1 Inr-containing promoter (wt Inr). Quantification of the experiments (n = 3) are shown in (B). The intensity of each transcription product was quantified using Image J software. Total pixels of each transcription product were measured and expressed as percentage related to the experiment without the addition of α-Amanitin, which was considered as 100% transcription.

Figure 3

Mutations in conserved positions impairs transcription initiation. Those conserved positions were changed to bases, as indicated at the top of the figure. It can be observed that mutations on conserved positions at −1, +1, +2, and +4 impair transcription initiation of the nmt1 Inr-containing promoter (indicated as wt).

Figure 4

Mediator depletion inhibits nmt1 Inr transcription. (A) Mediator was depleted from a WCE from a TAP-tagged Med7 strain using IgG-Sepharose beads, and transcription was recovered by replacing different combinations of factors. The WCE from the same TAP-tagged strain was depleted of TAFs using anti-TAF1 antibodies crosslinked to Sepharose beads. It is observed that Mediator depletion completely abolishes transcription of the nmt1 Inr (WCED), and is recovered by replacing an eluate from the IgG-Sepharose beads (WCED + IgG Seph. Elution), with a crude fraction from a P11 chromatographic fraction of WCE (WCED + 0.5 M P11), or with the same fraction plus the elution of the IgG-Sepharose column (WCED + IgG Seph + 0.5 M P11). TFIIB, TBP, RNAPII, or TFIIF cannot complement for the activity eluted from the IgG-Sepharose beads. Importantly, TAF-depletion has no effect on nmt1 Inr transcription (WCE TAP + αTAF1) and an eluate from the anti-TAF1 beads cannot complement Mediator depletion (WCED + TAF1). (B) Western blot analysis with different antibodies was used to test the extension of the depletion of the WCED. It is observed that Mediator was completely depleted (Med17 panel) by IgG Sepharose beads, and TAFs are depleted by anti-TAF1 Sepharose beads (TAF5 panel). Depletion of Mediator does not affect the levels of TAFs, and depletion of TAFs does not affect Mediator levels. Levels of RNAPII and other transcription factors are not affected by either Mediator- or TAF-depletion.

Figure 5

TBP binds to the nmt1 Inr in a complex, either with TFIIA or TFIIB. (A) TFIIA/TFIIB added together to a binding assay with a labeled probe containing the wild-type nmt1 Inr do not produce a detectable complex; however, when TFIIA or TFIIB are added together with TBP, complexes are obtained, indicating that TBP binds to the Inr in the presence of these factors. (B) Mutants of the nmt1 Inr, which are impaired in transcription, cannot form a TFIIB-TBP complex, indicating that these conserved positions are required for TFIIB-TBP complex formation. (C) Preinitiation complex formation on the nmt1 Inr follows a similar pathway as TATA box-containing promoters. It can be observed that TFIIB-TBP does not form a new complex with RNAPII in the absence of TFIIF; however, when RNAPII + TFIIF (RNAPIIIIF) are added, a new complex is formed that migrates slower than the TFIIB-TBP complex. Adding TFIIH to this complex does not produce any change in its mobility or amount; however, when TFIIE is added, the intensity of the complex augments, indicating that TFIIE was incorporated. A similar process occurs when TFIIH is added to the TFIIB-TBP-RNAPII-TFIIF-TFIIE complex. In the absence of TFIIB, RNAPII + TFIIF is not recruited into the complex, indicating that TFIIB makes a bridge between TBP and RNAPII + TFIIF. Figure 5C was constructed from two panels from the same Western blot.

Figure 6

Nmt1 Inr transcription can be reconstituted with GTFs, RNAPII, Mediator, PC4, and a crude fraction from the 0.5 M P11 chromatographic column. A transcription assay for the nmt1 Inr was set up with GTFs, RNAPII, Mediator, and PC4; however, this purified system was not able to transcribe the nmt1 promoter. Addition of a crude chromatographic fraction from a P11 column (0.5 M P11) can support transcription in this assay. Addition of other chromatographic fractions from the P11 column (0.1 M and 0.3 M P11) do not support transcription from this promoter. (A) Omission of Mediator from the assay slightly diminishes transcription. (B) Chromatographic scheme of purification of TICF through several steps. The activity was followed by transcription assays set up with the nmt1 Inr template, GTFs, RNAPII, Mediator, and PC4. (C) Transcription assay and estimation of the molecular weight of TICF by gel filtration (AcA22 column). The molecular weight of TICF is near 500 kDa, as determined in the gel filtration column. (D) The activity of TICF is dependent on PC4. A transcription assay set up with GTFs, Mediator, and RNAPII can transcribe the nmt1 Inr when TICF and PC4 are added; however, when PC4 is absent from the assay (-PC4 + TICF), transcription no longer occurs. Consequently, when TCIF is omitted (+PC4-TCIF), transcription does not occur. The crude chromatographic fraction of 0.5 M P11 can replace TICF and PC4 (+0.5 M P11), however, the 0.3 M P11 fraction cannot replace the activities (+0.5 M P11). Tx indicates the transcription product.

Figure 7

Inr of the hDHFR/nmt1 family can be transcribed in WCE. Three promoters, namely FP000624 (FP 624), FP000463 (FP 463), and FP000396) (FP 396) were cloned (from −20 to +10), fused to the G-less cassette, transcribed in WCE, and compared with the nmt1 Inr +1 mutant (nmt1Inr + 1mut) and the wild-type nmt1 Inr (nmt1Inr). It can be observed that the three promoters can support transcription initiation in crude extracts. Tx indicates the transcription product.