Warner, M. H. | bioRxiv Science

bioRxiv2025

Genetic dissection of transcription start site selection by RNA polymerase II in Saccharomyces cerevisiae

Transcription initiation by RNA Pol II is driven by a preinitiation complex (PIC) comprising Pol II and general transcription factors (GTFs): TFIIA, TFIIB, TFIID, TFIIE, TFIIF and TFIIH. In Saccharomyces cerevisiae, transcription start site (TSS) selection proceeds by a promoter scanning mechanism where the PIC scans downstream for TSSs. To determine which factors participate in TSS selection by promoter scanning, we designed and implemented forward genetic selections using initiation-sensitive genetic reporters. These reporters were of two types, either being sensitive to upstream or downstream TSS shifts, allowing detection of alleles that affect promoter scanning in different directions. From >1000 candidates, we identified three primary classes of mutants: existing and novel mutants within known PIC components, including novel alleles of multiple TFIIH subunits; mutants that alter promoter scanning indirectly through cellular conditions (GTP levels and Mn2+ levels); and mutants in chromatin and transcription elongation-related factors. Genome-wide analysis using TSS mapping shows that tested PIC mutants alter TSS usage globally across promoters while tested chromatin and transcription elongation mutants had much more limited effects. These studies expand the spectrum of mutants able to perturb initiation by promoter scanning and suggest potential plasticity in the TFIIH structure that may be part of the evolution of this initiation mechanism.

molecular biology↗

bioRxiv2019

The Paf1 complex broadly impacts the transcriptome of Saccharomyces cerevisiae

The Polymerase Associated Factor 1 complex (Paf1C) is a multifunctional regulator of eukaryotic gene expression important for the coordination of transcription with chromatin modification and post-transcriptional processes. In this study, we investigated the extent to which the functions of Paf1C combine to regulate the Saccharomyces cerevisiae transcriptome. While previous studies focused on the roles of Paf1C in controlling mRNA levels, here we took advantage of a genetic background that enriches for unstable transcripts and demonstrate that deletion of PAF1 affects all classes of Pol II transcripts including multiple classes of noncoding RNAs. By conducting a de novo differential expression analysis independent of gene annotations, we found that Paf1 positively and negatively regulates antisense transcription at multiple loci. Comparisons with nascent transcript data revealed that many, but not all, changes in RNA levels detected by our analysis are due to changes in transcription instead of post-transcriptional events. To investigate the mechanisms by which Paf1 regulates protein-coding genes, we focused on genes involved in iron and phosphate homeostasis, which were differentially affected by PAF1 deletion. Our results indicate that Paf1 stimulates phosphate gene expression through a mechanism that is independent of any individual Paf1C-dependent histone modification. In contrast, the inhibition of iron gene expression by Paf1 correlates with a defect in H3 K36 tri-methylation. Finally, we showed that one iron regulon gene, FET4, is coordinately controlled by Paf1 and transcription of upstream noncoding DNA. Together these data identify roles for Paf1C in controlling both coding and noncoding regions of the yeast genome.

genomics↗