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NC2 has a role in determining the transcription start site in Saccharomyces cerevisiae

ABSTRACT: In this study we show that disruption of NC2 function by shutting off either of its two protein subunits in yeast provokes an immediate increase in cryptic transcription around and inside any kind of RNA pol II genes.

ORGANISM(S): Saccharomyces cerevisiae

PROVIDER: GSE67267 | GEO | 2016/03/01

SECONDARY ACCESSION(S): PRJNA279389

REPOSITORIES: GEO

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Detection of cryptic start codons in set2D by 5PSeq

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Quantitative analysis of transcription start site selection in Saccharomyces cerevisiae reveals control by DNA sequence, RNA Polymerase II activity, and NTP levels

Project description:Transcription start site (TSS) selection is a key step in gene expression and occurs at many promoter positions over a wide range of efficiencies. Here, we develop a massively parallel reporter assay to quantitatively dissect contributions of promoter sequence, NTP substrate levels, and RNA polymerase II (Pol II) activity to TSS selection by "promoter scanning" in Saccharomyces cerevisiae (Pol II MAssively Systematic Transcript End Readout, "Pol II MASTER"). Using Pol II MASTER, we measure the efficiency of Pol II initiation at 1,000,000 individual TSS sequences in a defined promoter context. Pol II MASTER confirms proposed critical qualities of S. cerevisiae TSS -8, -1, and +1 positions quantitatively in a controlled promoter context. Pol II MASTER extends quantitative analysis to surrounding sequences and determines that they tune initiation over a wide range of efficiencies. These results enabled the development of a predictive model for initiation efficiency based on sequence. We show that genetic perturbation of Pol II catalytic activity alters initiation efficiency mostly independently of TSS sequence, but selectively modulates preference for initiating nucleotide. Intriguingly, we find that Pol II initiation efficiency is directly sensitive to GTP levels at the first five transcript positions and to CTP and UTP levels at the second position genome wide. These results suggest individual NTP levels can have transcript-specific effects on initiation, representing a cryptic layer of potential regulation at the level of Pol II biochemical properties. The results establish Pol II MASTER as a method for quantitative dissection of transcription initiation in eukaryotes.

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