Uncoupling the TFIIH Core and Kinase Modules Leads To Misregulated RNA Polymerase II CTD Serine 5 Phosphorylation
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eLife Assessment
This important work demonstrates the role of physically linking the core and CTD kinase modules of TFIIH via separate domains of subunit Tfb3 in confining RNA Polymerase II Serine 5 CTD phosphorylation to promoter regions of transcribed genes in budding yeast. The main findings, resulting from analyses of viable Tfb3 mutants in which the linkage between TFIIH core and kinase modules has been severed, are supported by solid evidence from in vitro and in vivo experiments. There is an intriguing possibility that the Tfb3-mediated connection between core and kinase modules of TFIIH is an evolutionary addition to an ancestral state of physically unconnected enzymes, which could be examined more rigorously with additional evolutionary analyses.
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Abstract
TFIIH is an essential transcription initiation factor for RNA polymerase II (RNApII). This multi-subunit complex comprises two modules that are physically linked by the subunit Tfb3 (MAT1 in metazoans). The Core Module, with two DNA-dependent ATPases and several additional subunits, promotes DNA unwinding. The Kinase Module phosphorylates the C-terminal domain (CTD) of RNApII subunit Rpb1, initiating a cycle of CTD modifications that coordinate exchange of initiation and elongation factors. Why these two disparate activities are bundled into one factor is not obvious, but the connection may provide temporal coordination during early initiation. When Tfb3 is split into two parts to uncouple the TFIIH modules, the resulting cells are viable but grow very slowly. Chromatin immunoprecipitation of the split TFIIH shows that the Core Module, but not the Kinase, is properly recruited to promoters. Instead of the normal promoter-proximal peak, high CTD Serine 5 phosphorylation is seen throughout transcribed regions. Therefore, coupling the TFIIH modules is necessary to localize and limit CTD kinase activity to early stages of transcription. These results are consistent with the idea that the two TFIIH modules began as independent functional entities that later became connected by Tfb3 during early eukaryotic evolution.
Impact Statement
The TFIIH subunit Tfb3/MAT1 can be split into two parts to uncouple the TFIIH kinase and DNA translocase modules, resulting in unfocused CTD phosphorylation.
Article activity feed
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eLife Assessment
This important work demonstrates the role of physically linking the core and CTD kinase modules of TFIIH via separate domains of subunit Tfb3 in confining RNA Polymerase II Serine 5 CTD phosphorylation to promoter regions of transcribed genes in budding yeast. The main findings, resulting from analyses of viable Tfb3 mutants in which the linkage between TFIIH core and kinase modules has been severed, are supported by solid evidence from in vitro and in vivo experiments. There is an intriguing possibility that the Tfb3-mediated connection between core and kinase modules of TFIIH is an evolutionary addition to an ancestral state of physically unconnected enzymes, which could be examined more rigorously with additional evolutionary analyses.
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Reviewer #1 (Public review):
Giordano et al. demonstrate that yeast cells expressing separated N- and C-terminal regions of Tfb3 are viable and grow well. Using this creative and powerful tool, the authors effectively uncouple CTD Ser5 phosphorylation at promoters and assess its impact on transcription. This strategy is complementary to previous approaches, such as Kin28 depletion or the use of CDK7 inhibitors. The results are largely consistent with earlier studies, reinforcing the importance of the Tfb3 linkage in mediating CTD Ser5 phosphorylation at promoters and subsequent transcription.
Notably, the authors also observe effects attributable to the Tfb3 linker itself, beyond its role as a simple physical connection between the N- and C-terminal domains. These findings provide functional insight into the Tfb3 linker, which had …
Reviewer #1 (Public review):
Giordano et al. demonstrate that yeast cells expressing separated N- and C-terminal regions of Tfb3 are viable and grow well. Using this creative and powerful tool, the authors effectively uncouple CTD Ser5 phosphorylation at promoters and assess its impact on transcription. This strategy is complementary to previous approaches, such as Kin28 depletion or the use of CDK7 inhibitors. The results are largely consistent with earlier studies, reinforcing the importance of the Tfb3 linkage in mediating CTD Ser5 phosphorylation at promoters and subsequent transcription.
Notably, the authors also observe effects attributable to the Tfb3 linker itself, beyond its role as a simple physical connection between the N- and C-terminal domains. These findings provide functional insight into the Tfb3 linker, which had previously been observed in structural studies but lacked clear functional relevance. Overall, I am very positive about this manuscript and offer a few minor comments below that may help to further strengthen the study.
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PIC structures show the linker emerging from the N-terminal domain as a long alpha-helix running along the interface between the two ATPase subunits, followed by a turn and a short stretch of helix just N-terminal to a disordered region that connects to the C-terminal region (see schematic in Figure 1A).
The linker helix was only observed in the poised PIC (Abril-Garrido et al., 2023), not in other fully-engaged PIC structures.
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Recent structures (reviewed in (Yu et al., 2023)) show that the Kinase Module would block interactions between the Core Module and other NER factors. Therefore, TFIIH either enters into the NER complex as the free Core Module, or the Kinase Module must dissociate soon after.
To my knowledge, this is still controversial in the NER field. I note the potential function of the kinase module is likely attributed to the N-terminal region of Tfb3 through its binding to Rad3. Because the yeast strains used in Figure 6 retain the N-terminal region of Tfb3, the UV sensitivity assay presented here is unlikely to directly address the contribution of the kinase module to NER.
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Notably, release of the Tfb3 Linker contact also results in the long alpha-helix becoming disordered (Abril-Garrido et al., 2023), which could allow the kinase access to a far larger radius of area. This flexibility could help the kinase reach both proximal and distal repeats within the CTD, which can theoretically extend quite far from the RNApII body.
Although the kinase module was resolved at low resolution in all PIC-Mediator structures, these structural studies consistently reveal the same overall positioning of the kinase module on Mediator, indicating that its localization is constrained rather than variable. This observation suggests that the linker region may help position the kinase module at this specific site, likely through direct interactions with the PIC or Mediator. This idea is further supported by numerous cross-links between the linker region and Mediator (Robinson et al., 2016).
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Reviewer #2 (Public review):
Summary:
This work advances our understanding of how TFIIH coordinates DNA melting and CTD phosphorylation during transcription initiation. The finding that untethered kinase activity becomes "unfocused," phosphorylating the CTD at ser5 throughout the coding sequence rather than being promoter-restricted, suggests that the TFIIH Core-Kinase linkage not only targets the kinase to promoters but also constrains its activity in a spatial and temporal manner.
Strengths:
The experiments presented are straightforward, and the models for coupling initiation and CTD phosphorylation and for the evolution of these linked processes are interesting and novel. The results have important implications for the regulation of initiation and CTD phosphorylation.
Weaknesses:
Additional data that should be easily obtainable and …
Reviewer #2 (Public review):
Summary:
This work advances our understanding of how TFIIH coordinates DNA melting and CTD phosphorylation during transcription initiation. The finding that untethered kinase activity becomes "unfocused," phosphorylating the CTD at ser5 throughout the coding sequence rather than being promoter-restricted, suggests that the TFIIH Core-Kinase linkage not only targets the kinase to promoters but also constrains its activity in a spatial and temporal manner.
Strengths:
The experiments presented are straightforward, and the models for coupling initiation and CTD phosphorylation and for the evolution of these linked processes are interesting and novel. The results have important implications for the regulation of initiation and CTD phosphorylation.
Weaknesses:
Additional data that should be easily obtainable and analysis of existing data would enable an additional test of the models presented and extract additional mechanistic insights.
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Reviewer #3 (Public review):
Summary:
Eukaryotic gene transcription requires a large assemblage of protein complexes that govern the molecular events required for RNA Polymerase II to produce mRNAs. One of these complexes, TFIIH, comprises two modules, one of which promotes DNA unwinding at promoters, while the other contains a kinase (Kin28 in yeast) that phosphorylates the repeated motif at the C-terminal domain (CTD) of the largest subunit of Pol II. Kin28 phosphorylation of Ser5 in the YSPTSPS motif of the CTD is normally highly localized at promoter regions, and marks the beginning of a cycle of phosphorylation events and accompanying protein association with the CTD during the transition from initiation to elongation.
The two modules of TFIIH are linked by Tfb3. Tfb3 consists of two globular regions, an N-terminal domain that …
Reviewer #3 (Public review):
Summary:
Eukaryotic gene transcription requires a large assemblage of protein complexes that govern the molecular events required for RNA Polymerase II to produce mRNAs. One of these complexes, TFIIH, comprises two modules, one of which promotes DNA unwinding at promoters, while the other contains a kinase (Kin28 in yeast) that phosphorylates the repeated motif at the C-terminal domain (CTD) of the largest subunit of Pol II. Kin28 phosphorylation of Ser5 in the YSPTSPS motif of the CTD is normally highly localized at promoter regions, and marks the beginning of a cycle of phosphorylation events and accompanying protein association with the CTD during the transition from initiation to elongation.
The two modules of TFIIH are linked by Tfb3. Tfb3 consists of two globular regions, an N-terminal domain that contacts the Core module of TFIIH and a C-terminal domain that contacts the kinase module, connected by a linker. In this paper, Giordano et al. test the role of Tfb3 as a connector between the two modules of TFIIH in yeast. They show that while no or very slow growth occurs if only the C-terminal or N-terminal region of Tfb3 is present, near normal growth is observed when the two unlinked regions are expressed. Consistent with this result, the separate domains are shown to interact with the two distinct TFIIH modules. ChIP experiments show that the Core module of TFIIH maintains its localization at gene promoters when the Tfb3 domains are separated, while localization of the kinase module and of Ser5 phosphorylation on the CTD of Pol II is disrupted. Finally, the authors examine the effect of separating the Tfb3 domains on another function of TFIIH, namely nucleotide excision repair, and find little or no effect when only the N-terminal region of Tfb3 or the two unlinked domains are present.
Strengths:
Experiments involving expression of Tfb3 domains in yeast are well-controlled, and the data regarding viability, interaction of the separate Tfb3 domains with TFIIH modules, genome-wide localization of the TFIIH modules and of phosphorylated Ser5 CTDs, and of effects on NER, are convincing. The experiments are consistent with current models of TFIIH structure and function and support a model in which Tfb3 tethers the kinase module of TFIIH close to initiation sites to prevent its promiscuous action on elongating Pol II.
Weaknesses:
(1) The work is limited in scope and does not provide any major insights into the mechanism of transcription. One indication of this limitation is that in the Discussion, published structural and functional results on transcription are used to support the interpretations of the results here more than current results inform previous models or findings.
(2) The first described experiment, which purports to show that three kinases cannot function in place of Kin28 when tethered (by fusion) to Tfb3, is missing the crucial control of showing that Kin28 can support viability in the same context. This result also does not connect with the rest of the manuscript.
(3) Finally, the authors present the interesting and reasonable speculation that the TFIIH complex and connecting Tfb3 found in mammals and yeast may have evolved from an earlier state in which the two TFIIH subdomains were present as unconnected, distinct enzymes. This idea is supported by a single example from the literature (T. brucei). A more thorough evolutionary analysis could have tested this idea more rigorously.
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