Ssl2/TFIIH function in transcription start site scanning by RNA Polymerase II in Saccharomyces cerevisiae

  1. Tingting Zhao
  2. Irina O Vvedenskaya
  3. William KM Lai
  4. Shrabani Basu
  5. B Franklin Pugh
  6. Bryce E Nickels
  7. Craig D Kaplan  Is a corresponding author
  1. University of Pittsburgh, United States
  2. Rutgers University, United States
  3. Cornell University, United States

Abstract

In Saccharomyces cerevisiae, RNA Polymerase II (Pol II) selects transcription start sites (TSS) by a unidirectional scanning process. During scanning, a preinitiation complex (PIC) assembled at an upstream core promoter initiates at select positions within a window ~40-120 basepairs downstream. Several lines of evidence indicate that Ssl2, the yeast homolog of XPB and an essential and conserved subunit of the general transcription factor (GTF) TFIIH, drives scanning through its DNA-dependent ATPase activity, therefore potentially controlling both scanning rate and scanning extent (processivity). To address questions of how Ssl2 functions in promoter scanning and interacts with other initiation activities, we leveraged distinct initiation-sensitive reporters to identify novel ssl2 alleles. These ssl2 alleles, many of which alter residues conserved from yeast to human, confer either upstream or downstream TSS shifts at the model promoter ADH1 and genome-wide. Specifically, tested ssl2 alleles alter TSS selection by increasing or narrowing the distribution of TSSs used at individual promoters. Genetic interactions of ssl2 alleles with other initiation factors are consistent with ssl2 allele classes functioning through increasing or decreasing scanning processivity but not necessarily scanning rate. These alleles underpin a residue interaction network that likely modulates Ssl2 activity and TFIIH function in promoter scanning. We propose that the outcome of promoter scanning is determined by two functional networks, the first being Pol II activity and factors that modulate it to determine initiation efficiency within a scanning window, and the second being Ssl2/TFIIH and factors that modulate scanning processivity to determine the width of the scanning widow.

Data availability

Genomics datasets generated in the current study are available in the NCBI BioProject and SRA, under the accession numbers of PRJNA681384 and SRP295731, respectively. The processed genomic data files are available in GEO, under the accession number of GSE182792. The streamlined commands to generate TSS-seq bedGraph files, count tables, tables of expression, spread and median TSS can be found at https://github.com/Kaplan-Lab-Pitt/Ssl2_scanning.

The following data sets were generated

Article and author information

Author details

  1. Tingting Zhao

    University of Pittsburgh, Pittsburgh, United States
    Competing interests
    No competing interests declared.
  2. Irina O Vvedenskaya

    Waksman Institute, Rutgers University, Piscataway, United States
    Competing interests
    No competing interests declared.
  3. William KM Lai

    Cornell University, Ithaca, United States
    Competing interests
    No competing interests declared.
  4. Shrabani Basu

    University of Pittsburgh, Pittsburgh, United States
    Competing interests
    No competing interests declared.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-5096-5490
  5. B Franklin Pugh

    Cornell University, Ithaca, United States
    Competing interests
    B Franklin Pugh, BFP has a financial interest in Peconic, LLC, which utilizes the ChIP-exo technology implemented in this study and could potentially benefit from the outcomes of this research..
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-8341-4476
  6. Bryce E Nickels

    Department of Genetics, Rutgers University, Piscataway, United States
    Competing interests
    No competing interests declared.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-7449-8831
  7. Craig D Kaplan

    University of Pittsburgh, Pittsburgh, United States
    For correspondence
    craig.kaplan@pitt.edu
    Competing interests
    No competing interests declared.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-7518-695X

Funding

National Institute of General Medical Sciences (R01GM120450)

  • Craig D Kaplan

National Institute of General Medical Sciences (R01GM059055)

  • B Franklin Pugh

National Institute of General Medical Sciences (R01GM059055)

  • Bryce E Nickels

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Reviewing Editor

  1. Eric J Wagner, University of Rochester Medical Center, United States

Publication history

  1. Preprint posted: May 5, 2021 (view preprint)
  2. Received: June 4, 2021
  3. Accepted: October 14, 2021
  4. Accepted Manuscript published: October 15, 2021 (version 1)
  5. Version of Record published: November 12, 2021 (version 2)

Copyright

© 2021, Zhao et al.

This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.

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  1. Tingting Zhao
  2. Irina O Vvedenskaya
  3. William KM Lai
  4. Shrabani Basu
  5. B Franklin Pugh
  6. Bryce E Nickels
  7. Craig D Kaplan
(2021)
Ssl2/TFIIH function in transcription start site scanning by RNA Polymerase II in Saccharomyces cerevisiae
eLife 10:e71013.
https://doi.org/10.7554/eLife.71013

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