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.

The Catalytic Subunit of Schizosaccharomyces pombe CK2 (Cka1) Negatively Regulates RNA Polymerase II Transcription through Phosphorylation of Positive Cofactor 4 (PC4)

“…Similar to budding yeast Sub1, fission yeast PC4 can bind single- and double-stranded DNA and it is able to mediate transcriptional activation together with TFIIA and Mediator [ 11 ]. Fission yeast PC4 is also required for in vitro transcription of ribosomal protein genes [ 50 ] and for transcription of promoters containing an Inr as the unique core promoter element [ 51 ]. However, fission yeast PC4 is not essential for cell viability (deletion mutant) and its absence does not cause any visible phenotype [ 52 ].…”

RNA Polymerase II Transcription in Pneumocystis: TFIIB from Pneumocystis carinii Can Replace the Transcriptional Functions of Fission Yeast Schizosaccharomyces pombe TFIIB In Vivo and In Vitro

“…EMSA assays were performed essentially as described previously [ 37 , 38 ]. Binding reactions contained binding mix: 20 mM HEPES (pH 7.9), 50 mM KCl, 5 mM MgCl 2 , 0.1 mM EDTA, 5% glycerol, 0.5% PEG 8000 (Sigma-Aldrich, St Louis, MO, USA), 2 mM DTT, 0.1 mM PMSF, 50 ng acetylated BSA and 50 ng poly(dG-dC).…”

“…In vitro transcription was performed as described previously [ 36 , 38 ] using 100 ng of plasmid DNA containing the Ad-MLP promoter fused to the G-less cassette. The plasmid DNA used for transcription reactions was purified by the E.Z.N.A.…”

Ino2, activator of yeast phospholipid biosynthetic genes, interacts with basal transcription factors TFIIA and Bdf1

“…The positive influence of TFIIA on TBP–TATA binding was shown to be particularly effective with a non-consensus TATA sequence (Stewart and Stargell 2001 ; Hieb et al 2007 ). In the yeast Schizosaccharomyces pombe , TFIIA + TBP (but not TBP alone) has been recently shown to bind to the initiator sequence of the strongly regulated nmt1 promoter (Rojas et al 2022 ). Human TFIIA not only supports TBP binding to the basal promoter but also interacts with a G-rich sequence motif closely upstream of the TATA element (designated IIARE, overlapping with the TFIIB recognition element, BRE u ; Wang et al 2017 ).…”