Ssl2/TFIIH function in transcription start site scanning by RNA polymerase II in Saccharomyces cerevisiae
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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.)
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Abstract
In Saccharomyces cerevisiae , RNA polymerase II (Pol II) selects transcription start sites (TSSs) 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 bp 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.
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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.)
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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 …
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.
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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 …
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.
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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 …
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.
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