Tfiih Generates a Six-Base-Pair Open Complex during Eukaryotic Transcription Initiation

Transcription initiation is a complex, energy consuming process that requires the massive and coordinated activity of many proteins often organized in big multi-subunit complexes. The General Transcription Factors (GTFs) and the TATA-binding protein (TBP) orchestrate the formation of the so-called pre-initiation complex (PIC), a key step of transcription initiation required for RNA Polymerase II (RNAPII) association to promoters. Although the PIC is made by the GTFs, many other complexes generally referred to as transcriptional co-activators participate in the formation of the PIC. Transcriptional co-activator complexes include chromatin remodelers, Histone AcetylTransferases (HATs) and the Mediator complex. In yeast, it is has been reported that two main HAT complexes, NuA4 and SAGA, and the Mediator complex have a global role in transcription. However, though, it is well-known that histone acetylation is associated to high transcription and that Mediator plays a central function in PIC assembly, the deployment of NuA4, SAGA and Mediator at yeast genes is still elusive. Here, we investigate the contribution of NuA4, SAGA and Mediator in RNAPII association at all yeast genes through rapid nuclear depletion of key complex subunits. We reveal that Gcn5, the HAT of the SAGA complex, is modestly but equally involved in transcription of all yeast genes, while Esa1, the HAT of the NuA4 complex, is most strongly required for transcription of certain groups of genes. Curiously, we show that nuclear depletion of Med17, an essential subunit of the Mediator complex, strongly affects transcription of a small subset of genes characterized by a well-conserved TATA box, highlighting a previously underappreciated connection between Mediator complex and TBP. Our analysis reveals that three combinations of co-activator deployment are used to generate high transcription levels and that transcription of two groups of so called “house-keeping” or “growth-promoting genes” is particularly dependent on Tra1, a shared component of NuA4 and SAGA. Our work on Ribosomal Protein (RP) gene and Ribosome Biogenesis (RiBi) gene transcription discloses the importance of the transcription factors Ifh1 and Sfp1 in recruiting Tra1 at these promoters. 10 Furthermore, a deep investigation of transcription factors binding at RP gene promoters highlights the existence of a hierarchy, with Rap1 being required for the association of the other TFs. Our study also reveals that RP gene promoters, as well as the promoters of many other highly transcribed genes, are characterized by the presence of one or more unusually unstable MNase-sensitive nucleosomes referred to as fragile nucleosomes. Furthermore, our work discovers the main role of Rap1 and of other pioneer transcription factors in establishing nucleosome fragility and promoter architecture. Finally, we investigate the biophysical properties of fragile nucleosomes and provide evidence of their nucleosomal nature. Altogether the work presented in this thesis elucidates important features of yeast promoters and provides new insights into the mechanisms that drive eukaryotic transcription.

Antibodies Directed against an Epitope in the N-Terminal Region of the H4L Subunit of the Vaccinia Virus RNA Polymerase Inhibit Both Transcription Initiation and Transcription Termination, in Vitro

Genetic studies in yeast have shown that the translation initiation factor eIF5 plays an important role in the selection of the AUG start codon. In order to ensure translation fidelity, the hydrolysis of GTP bound to the 40S preinitiation complex (40S.Met-tRNA(i).eIF2.GTP), promoted by eIF5, must occur only when the complex has selected the AUG start codon. However, the mechanism that prevents the eIF5-promoted GTP hydrolysis, prior to AUG selection by the ribosomal machinery, is not known. In this work, we show that the presence of initiation factors eIF1, eIF1A and eIF3 in the 40S preinitiation complex (40S.eIF1.eIF1A.eIF3.Met-tRNA(i).eIF2.GTP) and the subsequent binding of the preinitiation complex to eIF4F bound at the 5'-cap structure of mRNA are necessary for preventing eIF5-promoted hydrolysis of GTP in the 40S preinitiation complex. This block in GTP hydrolysis is released upon AUG selection by the 40S preinitiation complex. These results, taken together, demonstrate the biochemical requirements for regulation of GTP hydrolysis and its coupling to the AUG selection process during translation initiation.

In vitro transcription was reconstituted with HeLa cell transcription factors and RNA polymerase II, which were essentially free from DNA topoisomerase activities. DNA templates with defined negative superhelical densities were tested for transcription activity. Transcription of the Bombyx mori fibroin gene increases and plateaus from templates of increasing superhelicity, and transcription from the adenovirus 2 major late promoter rises and then falls, while transcription of the Drosophila hsp70 gene remains unchanged. Dissection of transcription into pre and post-initiation steps by the use of Sarkosyl reveals that formation of a preinitiation complex on the fibroin gene or the adenovirus 2 major late promoter is slow on relaxed DNA and accelerated by DNA superhelicity. On the contrary, the preinitiation complex assembles rapidly on the hsp70 gene irrespective of DNA topology. As is the case with the fibroin gene promoter, DNA superhelicity appears to facilitate the interaction of transcription factor IID to the adenovirus 2 major late promoter.

In vitro reconstitution of yeast RNA polymerase II transcription initiation with high efficiency.

DNA supercoiling facilitates the assembly of transcriptionally active chromatin on the adenovirus major late promoter

BackgroundProteins in their majority act rarely as single entities. Multisubunit macromolecular complexes are the actors in most of the cellular processes. These nanomachines are hold together by weak protein-protein interactions and undergo functionally important conformational changes. TFIID is such a multiprotein complex acting in eukaryotic transcription initiation. This complex is first to be recruited to the promoter of the genes and triggers the formation of the transcription preinitiation complex involving RNA polymerase II which leads to gene transcription. The exact role of TFIID in this process is not yet understood.MethodsLast generation electron microscopes, improved data collection and new image analysis tools made it possible to obtain structural information of biological molecules at atomic resolution. Cryo-electron microscopy of vitrified samples visualizes proteins in a fully hydrated, close to native state. Molecular images are recorded at liquid nitrogen temperature in low electron dose conditions to reduce radiation damage. Digital image analysis of these noisy images aims at improving the signal-to-noise ratio, at separating distinct molecular views and at reconstructing a three-dimensional model of the biological particle.ResultsUsing these methods we showed the early events of an activated transcription initiation process. We explored the interaction of the TFIID coactivator with the yeast Rap1 activator, the transcription factor TFIIA and the promoter DNA. We demonstrated that TFIID serves as an assembly platform for transient protein-protein interactions, which are essential for transcription initiation.ConclusionsRecent developments in electron microscopy have provided new insights into the structural organization and the dynamic reorganization of large macromolecular complexes. Examples of near-atomic resolutions exist but the molecular flexibility of macromolecular complexes remains the limiting factor in most case. Electron microscopy has the potential to provide both structural and dynamic information of biological assemblies in order to understand the molecular mechanisms of their functions.

Promoter recognition is the first and the most important step during gene expression. Our studies of the yeast (Saccharomyces cerevisiae) mitochondrial (mt) transcription machinery provide mechanistic understandings on the basic problem of how the mt RNA polymerase (RNAP) with the help of the initiation factor discriminates between promoter and non-promoter sequences. We have used fluorescence-based approaches to quantify DNA binding, bending, and opening steps by the core mtRNAP subunit (Rpo41) and the transcription factor (Mtf1). Our results show that promoter recognition is not achieved by tight and selective binding to the promoter sequence. Instead, promoter recognition is achieved by an induced-fit mechanism of transcription factor-dependent differential conformational changes in the promoter and non-promoter DNAs. While Rpo41 induces a slight bend upon binding both the DNAs, addition of the Mtf1 results in severe bending of the promoter and unbending of the non-promoter DNA. Only the sharply bent DNA results in the catalytically active open complex. Such an induced-fit mechanism serves three purposes: 1) assures catalysis at promoter sites, 2) prevents RNA synthesis at non-promoter sites, and 3) provides a conformational state at the non-promoter sites that would aid in facile translocation to scan for specific sites.

We have used alanine scanning to analyze protein-protein interactions by human TATA-element binding protein (TBP) within the transcription preinitiation complex. The results indicate that TBP interacts with RNA polymerase II and general transcription factors IIA, IIB, and IIF within the functional transcription preinitiation complex and define the determinants of TBP for each of these interactions. The results permit construction of a model for the structure of the preinitiation complex.

Only a small proportion (<2%) of RNA polymerase I (pol I) from whole-cell extracts appeared to be competent for specific initiation at the ribosomal gene promoter in a yeast reconstituted transcription system. Initiation-competent pol I molecules were found exclusively in salt-resistant complexes that contain the pol I-specific initiation factor Rrn3p. Levels of initiation-competent complexes in extracts were independent of total Rrn3p content and varied with the growth state of the cells. Although extracts from stationary phase cells contained substantial amounts of Rrn3p and pol I, they lacked the pol I-Rrn3p complex and were inactive in promoter-dependent transcription. Activity was restored by adding purified pol I-Rrn3p complex to extracts from stationary phase cells. The pol I-Rrn3p complex dissociated during transcription and lost its capacity for subsequent reinitiation in vitro, suggesting a stoichiometric rather than a catalytic activity in initiation. We propose that the formation and disruption of the pol I-Rrn3p complex reflects a molecular switch for regulating rRNA synthesis and its growth rate-dependent regulation.

Translation initiation is the rate‐limiting and most‐regulated phase of translation, and its misregulation has been implicated in myriad maladies, including cancer and neurodegenerative disease. In eukaryotes, initiation is a complex pathway requiring the participation of at least twelve initiation factors (eIFs). Eukaryotic translation initiation factor 3 (eIF3) is the largest and most complex of these factors and appears to participate in events throughout the pathway.In particular, eIF3 is a pivotal player in the recruitment of mRNA to the translation pre‐initiation complex (PIC). Biochemical and genetic studies have identified interactions of eIF3 with the PIC near the mRNA entry and exit channels of the ribosome; structural studies have revealed that eIF3 binds the solvent face of the small (40S) ribosomal subunit, projecting arms into both mRNA channels and ultimately encircling the PIC.We recently probed the effects of a library of S. cerevisiae eIF3 functional variants using an in‐vitro reconstituted system. These experiments demonstrated that mutations throughout eIF3 disrupt its interaction with the PIC and diminish its ability to accelerate recruitment of a native yeast mRNA. Using a series of model mRNAs to control the amount of nucleotide sequence in either the mRNA entry or exit channel, we further revealed a critical role for eIF3 in stabilizing mRNA at the exit channel. Together, these experiments are consistent with the entry‐ and exit‐channel arms of eIF3 playing distinct roles during PIC formation, mRNA recruitment, and scanning.We have also investigated the collaboration of eIF3 with Rps3/uS3 in stabilizing mRNA within the mRNA entry channel. We found that Rps3/uS3 residues previously implicated in the helicase activity of the elongating ribosome stabilize mRNA binding within the ribosomal entry channel and enhance the effects of eIF3 on mRNA binding in both channels. More recently, we have observed that eIF3i and eIF3g contribute to the PIC‐dependent enhancement of eIF4A ATPase activity, which recent work suggests may function to remodel the mRNA entry channel.Our current work is focused on dissecting the interactions between eIF3, the PIC, and other initiation factors in both the mRNA entry and exit channels, as well as on the inter‐subunit face of the PIC, using both in‐vitro and next‐generation sequencing approaches. In particular, we are exploring the genome‐wide translational effects of depleting or disrupting the eIF3 complex, using ribosome profiling. In parallel, we are investigating the effects of mutations to the 40S protein Rps2 on the PIC:mRNA interaction, as well as how mutations to Rps2 and Rps3 affect the sensitivity of this interaction to mutations within the eIF3 complex.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

TFIIF is considered to be a general transcription factor, based on the fact that it is essential for assembly of RNA polymerase II preinitiation complexes on fully double-stranded templates in vitro. Existing models assign various tasks to TFIIF during preinitiation complex formation and transcript initiation. Recent results do not support all aspects of those models but they do emphasize the significance of the interaction of TFIIF and TFIIB. Other recent findings raise the possibility that a fraction of RNA polymerase II transcription complex assembly proceeds through a pathway that is independent of TFIIF.

Head Module Control of Mediator Interactions

Dynamic Architecture of a Minimal RNA Polymerase II Open Promoter Complex

The transcriptional regulator Taf14 is a component of multiple protein complexes involved in transcription initiation and chromatin remodeling in yeast cells. Although Taf14 is not required for cell viability, it becomes essential in conditions where the formation of the transcription preinitiation complex is hampered. The specific role of Taf14 in mediating transcription initiation and preinitiation complex formation is unclear. Here, we explored its role in the general transcription factor IID by mapping Taf14 genetic and proteomic interactions and found that it was needed for the function of the complex if Htz1, the yeast homolog of histone H2A.Z, was absent from chromatin. Dissecting the functional domains of Taf14 revealed that the linker region between the YEATS and ET domains was required for cell viability in the absence of Htz1 protein. We further show that the linker region of Taf14 interacts with DNA. We propose that providing additional DNA binding capacity might be a general role of Taf14 in the recruitment of protein complexes to DNA and chromatin.