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  1. Version 3 published on bioRxiv
  2. eLife

    Evaluation Summary:

    Kaplan and colleagues build upon their earlier work using genetic phenotypes to find and analyze mutations that determine how mRNA start sites are chosen. Here they provide convincing genetic evidence supporting a model in which the Transcription Factor II H (TFIIH) protein complex pushes downstream DNA back into the RNA polymerase active site, creating a window within which the polymerase can choose particular start sites. This will primarily interest those in the transcription field who are thinking about initiation mechanisms.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)

  3. eLife

    Reviewer #1 (Public Review):

    The goal of this study was to generate and analyze phenotypes associated with alleles of ssl2 that alter transcription start site (TSS) selection. Specifically, the authors created a screening method to detect either upstream TSS shifts or downstream distinctly and then use this approach to create new alleles of ssl2 that might reveal important residues/regions of ssl2 involved in promoter scanning. To that end, new alleles were not only characterized using reporter-based methods but were broadened to genome-wide analysis through a transcriptional start site sequencing approach. Ssl2 alleles were further characterized in combination with Pol II alleles that also impact TSS selection to build a framework of additive and epistatic interactions. Overall, this study was conducted in a very thorough manner and is likely to make an impact on the field by provided insight as to how the key enzymatic players work to initiate transcription. The primary weakness of the paper is that of presentation as the authors attention to detail at times was very dense and thus reduced readability of the study.

  4. eLife

    Reviewer #2 (Public Review):

    This work is based on the use of multiple reporter systems for TSS usage based on yeast growth (Fig 1A). Those reporters enable to use cell growth as a proxy for changes in TSS usage.

    The authors combine the use those systems and with primer extension assays to screen a big number of known and novel mutants. Using complementary screen strategies, they screen for novel mutants of sll2 associated with changes in TSS usage. The authors analyze the distribution of the identified mutations and focus on 2 groups of alleles conferring upstream or downstream TSS shifts. Then, they confirm for selected mutants that the identified phenotypes are indeed associated to genome-wide changes in the distribution of TSS. Next, they investigate the genetic interaction between the new sll2 alleles and known mutants able to change TSS that the authors have previously characterized. Finally, the authors show that the changes in TSS are also associated with subtle differences in PIC position as measured by ChIP-Exo. This is a strong work based on meticulous use of yeast genetics and confirmed by direct measure of the molecular phenotypes by TSS-Seq and ChIP-Exo. By its own nature, the presented work is complex and difficult to follow. Despite that, the authors include multiple schemes to facilitate its interpretation.

  5. eLife

    Reviewer #3 (Public Review):

    Biochemical and structural work has produced a model in which TFIIH translocase pushes downstream DNA back towards the active site to create a transcription bubble. In most eukaryotes, the transcription start site (TSS) appears within this initial bubble at a relatively fixed distance from the TATA box. However, at Saccharomyces cerevisiae promoters, multiple TSSs are spread in a window over a larger range. To explain this, a "scanning" mechanism has been proposed. Earlier work from the Kaplan lab and others provides support for directional scanning, where individual TSSs each have a certain probability of functioning. The Kaplan lab has shown that mutations in the RNA pol II active site can shift TSS choice upstream or downstream, with polymerases having faster elongation rates tending to choose upstream TSSs, while slower polymerases start further downstream. Here, Kaplan and colleagues extend their analysis to TFIIH mutants, providing genetic evidence that TFIIH function (and presumably translocation rate and/or processivity) is also a determinant of TSS choice. The one major weakness of the paper is that the TFIIH mutants are not tested biochemically to determine their actual defects (i.e. translocation rate, processivity, etc.), but I think that's a major undertaking that would be outside the scope of this paper. Based on genetic interactions between mutant alleles of TFIIH and other PIC components, a plausible and reasonably persuasive model is proposed for how the initiation/polymerization rate determines TSS choice within a window set by TFIIH translocase processivity.

  6. Version 2 published on bioRxiv
  7. Version 1 published on bioRxiv