Experimental and bioinformatic studies of transcription initiation by RNA polymerase II (RNAP2) have revealed a mechanism of RNAP2 transcription initiation less uniform across gene promoters than initially thought. However, the general transcription factor TFIIB is presumed to be universally required for RNAP2 transcription initiation. Based on bioinformatic analysis of data and effects of TFIIB knockdown in primary and transformed cell lines on cellular functionality and global gene expression, we report that TFIIB is dispensable for transcription of many human promoters, but is essential for herpes simplex virus-1 (HSV-1) gene transcription and replication. We report a novel cell cycle TFIIB regulation and localization of the acetylated TFIIB variant on the transcriptionally silent mitotic chromatids. Taken together, these results establish a new paradigm for TFIIB functionality in human gene expression, which when downregulated has potent anti-viral effects.

Transcription of protein-encoding genes in eukaryotic cells requires the coordinated action of multiple general transcription factors (GTFs) and RNA polymerase II (Pol II). A "step-wise" preinitiation complex (PIC) assembly model has been suggested based on conventional ensemble biochemical measurements, in which protein factors bind stably to the promoter DNA sequentially to build a functional PIC. However, recent dynamic measurements in live cells suggest that transcription factors mostly interact with chromatin DNA rather transiently. To gain a clearer dynamic picture of PIC assembly, we established an integrated in vitro single-molecule transcription platform reconstituted from highly purified human transcription factors and complemented it by live-cell imaging. Here we performed real-time measurements of the hierarchal promoter-specific binding of TFIID, TFIIA, and TFIIB. Surprisingly, we found that while promoter binding of TFIID and TFIIA is stable, promoter binding by TFIIB is highly transient and dynamic (with an average residence time of 1.5 sec). Stable TFIIB-promoter association and progression beyond this apparent PIC assembly checkpoint control occurs only in the presence of Pol II-TFIIF. This transient-to-stable transition of TFIIB-binding dynamics has gone undetected previously and underscores the advantages of single-molecule assays for revealing the dynamic nature of complex biological reactions.

Transcription in eukaryotes is a multistep process involving the assembly and disassembly of numerous inter- and intramolecular interactions between transcription factors and nucleic acids. The roles of each of these interactions and the regions responsible for them have been identified and studied primarily by the use of mutants, which destroy the inherent properties of the interacting surface. A less intrusive but potentially effective way to study the interactions as well as the surfaces responsible for them is the use of RNA aptamers that bind to the interacting factors. Here, we report the isolation and characterization of high-affinity RNA aptamers that bind to the yeast general transcription factor TFIIB. These aptamers fall into two classes that interfere with TFIIB's interactions with either TBP or RNA polymerase II, both of which are crucial for transcription in yeast. We demonstrate the high affinity and specificity of these reagents, their effect on transcription and preinitiation complex formation and discuss their potential use to address mechanistic questions in vitro as well as in vivo.

Rpc82 is a TFIIE-related subunit of the eukaryotic RNA polymerase III (pol III) complex. Rpc82 contains four winged-helix (WH) domains and a C-terminal coiled-coil domain. Structural resolution of the pol III complex indicated that Rpc82 anchors on the clamp domain of the pol III cleft to interact with the duplex DNA downstream of the transcription bubble. However, whether Rpc82 interacts with a transcription factor is still not known. Here, we report that a structurally disordered insertion in the third WH domain of Rpc82 is important for cell growth and in vitro transcription activity. Site-specific photo-crosslinking analysis indicated that the WH3 insertion interacts with the TFIIB-related transcription factor Brf1 within the pre-initiation complex (PIC). Moreover, crosslinking and hydroxyl radical probing analyses revealed Rpc82 interactions with the upstream DNA and the protrusion and wall domains of the pol III cleft. Our genetic and biochemical analyses thus provide new molecular insights into the function of Rpc82 in pol III transcription.

TFIIB-related factor 2 (Brf2) is a member of the family of TFIIB-like core transcription factors. Brf2 recruits RNA polymerase (Pol) III to type III gene-external promoters, including the U6 spliceosomal RNA and selenocysteine tRNA genes. Found only in vertebrates, Brf2 has been linked to tumorigenesis but the underlying mechanisms remain elusive. We have solved crystal structures of a human Brf2-TBP complex bound to natural promoters, obtaining a detailed view of the molecular interactions occurring at Brf2-dependent Pol III promoters and highlighting the general structural and functional conservation of human Pol II and Pol III pre-initiation complexes. Surprisingly, our structural and functional studies unravel a Brf2 redox-sensing module capable of specifically regulating Pol III transcriptional output in living cells. Furthermore, we establish Brf2 as a central redox-sensing transcription factor involved in the oxidative stress pathway and provide a mechanistic model for Brf2 genetic activation in lung and breast cancer.

RNA polymerase III (RNA pol III) transcribes many of the small structural RNA molecules involved in processing and translation, thereby regulating the growth rate of a cell. Initiation of pol III transcription requires the evolutionarily conserved pol III initiation factor TFIIIB. TFIIIB is the molecular target of regulation by tumor suppressors, including p53, RB and the RB-related pocket proteins. However, our understanding of negative regulation of human TFIIIB-mediated transcription by other proteins is limited. In this study we characterize a RNA pol III luciferase assay and further demonstrate in vivo that a human homolog of yeast Maf1 represses RNA pol III transcription. Additionally, we show that Maf1 repression of RNA pol III transcription occurs via TFIIIB, specifically through the TFIIB family members Brf1 and Brf2.

Although TFIIB is widely regarded as an initiation factor, recent reports have implicated it in multiple aspects of eukaryotic transcription. To investigate the broader role of TFIIB in transcription, we performed quantitative proteomic analysis of yeast TFIIB. We purified two different populations of TFIIB; one from soluble cell lysate, which is not engaged in transcription, and the other from the chromatin fraction which yields the transcriptionally active form of the protein. TFIIB purified from the chromatin exhibits several interactions that explain its non-canonical roles in transcription. RNAPII, TFIIF and TFIIH were the only components of the preinitiation complex with a significant presence in chromatin TFIIB. A notable feature was enrichment of all subunits of CF1 and Rat1 3' end processing-termination complexes in chromatin-TFIIB preparation. Subunits of the CPF termination complex were also detected in both chromatin and soluble derived TFIIB preparations. These results may explain the presence of TFIIB at the 3' end of genes during transcription as well as its role in promoter-termination interaction.

Viral infections and the harm they cause to their host are a perpetual threat to living organisms. Pathogenesis and subsequent spread of infection requires replication of the viral genome and expression of structural and non-structural proteins of the virus. Generally, viruses use transcription and translation machinery of the host cell to achieve this objective. The viral genome encodes transcriptional regulators that alter the expression of viral and host genes by manipulating initiation and termination steps of transcription. The regulation of the initiation step is often through interactions of viral factors with gene specific factors as well as general transcription factors (GTFs). Among the GTFs, TFIIB (Transcription Factor IIB) is a frequent target during viral pathogenesis. TFIIB is utilized by a plethora of viruses including human immunodeficiency virus, herpes simplex virus, vaccinia virus, Thogoto virus, hepatitis virus, Epstein-Barr virus and gammaherpesviruses to alter gene expression. A number of viral transcriptional regulators exhibit a direct interaction with host TFIIB in order to accomplish expression of their genes and to repress host transcription. Some viruses have evolved proteins with a three-dimensional structure very similar to TFIIB, demonstrating the importance of TFIIB for viral persistence. Upon viral infection, host transcription is selectively altered with viral transcription benefitting. The nature of viral utilization of TFIIB for expression of its own genes, along with selective repression of host antiviral genes and downregulation of general host transcription, makes TFIIB a potential candidate for antiviral therapies.

Negative co-factor 2 (NC2) is a conserved eukaryotic complex composed of two subunits, NC2alpha (Drap1) and NC2beta (Dr1) that associate through a histone-fold motif. In this work, we generated mutants of NC2, characterized target genes for these mutants and studied the assembly of NC2 and general transcription factors on target promoters. We determined that the two NC2 subunits mostly function together to be recruited to DNA and regulate gene expression. We found that NC2 strongly controls promoter association of TFIIB, both negatively and positively. We could attribute the gene-specific repressor effect of NC2 on TFIIB to the C-terminal domain of NC2beta, and define that it requires ORF sequences of the target gene. In contrast, the positive function of NC2 on TFIIB targets is more general and requires adequate levels of the NC2 histone-fold heterodimer on promoters. Finally, we determined that NC2 becomes limiting for TATA-binding protein (TBP) association with a heat inducible promoter under heat stress. This study demonstrates an important positive role of NC2 for formation of the pre-initiation complex on promoters, under normal conditions through control of TFIIB, or upon activation by stress via control of TBP.

Structural comparisons of initiating RNA polymerase complexes and structure-based amino acid sequence alignments of general transcription initiation factors (eukaryotic TFIIB, archaeal TFB and bacterial σ factors) show that these proteins are homologs. TFIIB and TFB each have two-five-helix cyclin-like repeats (CLRs) that include a C-terminal helix-turn-helix (HTH) motif (CLR/HTH domains). Four homologous HTH motifs are present in bacterial σ factors that are relics of CLR/HTH domains. Sequence similarities clarify models for σ factor and TFB/TFIIB evolution and function and suggest models for promoter evolution. Commitment to alternate modes for transcription initiation appears to be a major driver of the divergence of bacteria and archaea.

Transcription initiation by archaeal RNA polymerase (RNAP) and eukaryotic RNAP II requires the general transcription factor (TF) B/ IIB. Structural analyses of eukaryotic transcription initiation complexes locate the B-reader domain of TFIIB in close proximity to the active site of RNAP II. Here, we present the first crosslinking mapping data that describe the dynamic transitions of an archaeal TFB to provide evidence for structural rearrangements within the transcription complex during transition from initiation to early elongation phase of transcription. Using a highly specific UV-inducible crosslinking system based on the unnatural amino acid para-benzoyl-phenylalanine allowed us to analyze contacts of the Pyrococcus furiosus TFB B-reader domain with site-specific radiolabeled DNA templates in preinitiation and initially transcribing complexes. Crosslink reactions at different initiation steps demonstrate interactions of TFB with DNA at registers +6 to +14, and reduced contacts at +15, with structural transitions of the B-reader domain detected at register +10. Our data suggest that the B-reader domain of TFB interacts with nascent RNA at register +6 and +8 and it is displaced from the transcribed-strand during the transition from +9 to +10, followed by the collapse of the transcription bubble and release of TFB from register +15 onwards.

Viruses have evolved a plethora of mechanisms to target host antiviral responses. Here, we propose a yet uncharacterized mechanism of immune regulation by the orthomyxovirus Thogoto virus (THOV) ML protein through engaging general transcription factor TFIIB. ML generates a TFIIB depleted nuclear environment by re-localizing it into the cytoplasm. Although a broad effect on gene expression would be anticipated, ML expression, delivery of an ML-derived functional domain or experimental depletion of TFIIB only leads to altered expression of a limited number of genes. Our data indicate that TFIIB is critically important for the de novo recruitment of Pol II to promoter start sites and that TFIIB may not be required for regulated gene expression from paused promoters. Since many immune genes require de novo recruitment of Pol II, targeting of TFIIB by THOV represents a neat mechanism to affect immune responses while keeping other cellular transcriptional activities intact. Thus, interference with TFIIB activity may be a favourable site for therapeutic intervention to control undesirable inflammation.

The effects of rapid acute depletion of components of RNA polymerase II (Pol II) general transcription factors (GTFs) that are thought to be critical for formation of preinitiation complexes (PICs) and initiation in vitro were quantified in HAP1 cells using precision nuclear run-on sequencing (PRO-Seq). The average dependencies for each factor across >70 000 promoters varied widely even though levels of depletions were similar. Some of the effects could be attributed to the presence or absence of core promoter elements such as the upstream TBP-specificity motif or downstream G-rich sequences, but some dependencies anti-correlated with such sequences. While depletion of TBP had a large effect on most Pol III promoters only a small fraction of Pol II promoters were similarly affected. TFIIB depletion had the largest general effect on Pol II and also correlated with apparent termination defects downstream of genes. Our results demonstrate that promoter activity is combinatorially influenced by recruitment of TFIID and sequence-specific transcription factors. They also suggest that interaction of the preinitiation complex (PIC) with nucleosomes can affect activity and that recruitment of TFIID containing TBP only plays a positive role at a subset of promoters.

The RNA polymerase (pol) II general transcription factor TFIIF functions at several steps in transcription initiation including preinitiation complex (PIC) formation and start site selection. We find that two structured TFIIF domains bind Pol II at separate locations far from the active site with the TFIIF dimerization domain on the Pol II lobe and the winged helix domain of the TFIIF small subunit Tfg2 above the Pol II protrusion where it may interact with upstream promoter DNA. Binding of the winged helix to the protrusion is PIC specific. Anchoring of these two structured TFIIF domains at separate sites locates an essential and unstructured region of Tfg2 near the Pol II active site cleft where it may interact with flexible regions of Pol II and the general factor TFIIB to promote initiation and start site selection. Consistent with this mechanism, mutations far from the enzyme active site, which alter the binding of either structured TFIIF domains to Pol II, have similar defects in transcription start site usage.

RNA polymerase II (Pol II) and its general transcription factors assemble on the promoters of mRNA genes to form large macromolecular complexes that initiate transcription in a regulated manner. During early transcription, these complexes undergo dynamic rearrangement and disassembly as Pol II moves away from the start site of transcription and transitions into elongation. One step in disassembly is the release of the general transcription factor TFIIB, although the mechanism of release and its relationship to the activity of transcribing Pol II is not understood. We developed a single-molecule fluorescence transcription system to investigate TFIIB release in vitro. Leveraging our ability to distinguish active from inactive complexes, we found that nearly all transcriptionally active complexes release TFIIB during early transcription. Release is not dependent on the contacts TFIIB makes with its recognition element in promoter DNA. We identified two different points in early transcription at which release is triggered, reflecting heterogeneity across the population of actively transcribing complexes. TFIIB releases after both trigger points with similar kinetics, suggesting the rate of release is independent of the molecular transformations that prompt release. Together our data support the model that TFIIB release is important for Pol II to successfully escape the promoter as initiating complexes transition into elongation complexes.

Specificity protein 1 (Sp1) is an important transcription factor implicated in numerous cellular processes. However, whether Sp1 is involved in the regulation of RNA polymerase III (Pol III)-directed gene transcription in human cells remains unknown. Here, we first show that filamin A (FLNA) represses Sp1 expression as well as expression of TFIIB-related factor 1 (BRF1) and general transcription factor III C subunit 2 (GTF3C2) in HeLa, 293T, and SaOS2 cell lines stably expressing FLNA-silencing shRNAs. Both BRF1 promoter 4 (BRF1P4) and GTF3C2 promoter 2 (GTF3C2P2) contain putative Sp1-binding sites, suggesting that Sp1 affects Pol III gene transcription by regulating BRF1 and GTF3C2 expression. We demonstrate that Sp1 knockdown inhibits Pol III gene transcription, BRF1 and GTF3C2 expression, and the proliferation of 293T and HeLa cells, whereas Sp1 overexpression enhances these activities. We obtained a comparable result in a cell line in which both FLNA and Sp1 were depleted. These results indicate that Sp1 is involved in the regulation of Pol III gene transcription independently of FLNA expression. Reporter gene assays showed that alteration of Sp1 expression affects BRF1P4 and GTF3C2P2 activation, suggesting that Sp1 modulates Pol III-mediated gene transcription by controlling BRF1 and GTF3C2 gene expression. Further analysis revealed that Sp1 interacts with and thereby promotes the occupancies of TATA box-binding protein, TFIIAα, and p300 at both BRF1P4 and GTF3C2P2. These findings indicate that Sp1 controls Pol III-directed transcription and shed light on how Sp1 regulates cancer cell proliferation.

RNA polymerase III (pol III) products play fundamental roles in a variety of cellular processes, including protein synthesis and cancer cell proliferation. In addition, dysregulation of pol III-directed transcription closely correlates with tumorigenesis. It is therefore of interest to identify novel pathways or factors governing pol III-directed transcription. Here, we show that transcription factor (TF) GATA binding protein 4 (GATA4) expression in SaOS2 cells was stimulated by the silencing of filamin A (FLNA), a repressor of pol III-directed transcription, suggesting that GATA4 is potentially associated with the regulation of pol III-directed transcription. Indeed, we show that GATA4 expression positively correlates with pol III-mediated transcription and tumor cell proliferation. Mechanistically, we found that GATA4 depletion inhibits the occupancies of the pol III transcription machinery factors at the loci of pol III target genes by reducing expression of both TFIIIB subunit TFIIB-related factor 1 and TFIIIC subunit general transcription factor 3C subunit 2 (GTF3C2). GATA4 has been shown to activate specificity factor 1 (Sp1) gene transcription by binding to the Sp1 gene promoter, and Sp1 has been confirmed to activate pol III gene transcription by directly binding to both Brf1 and Gtf3c2 gene promoters. Thus, the findings from this study suggest that GATA4 links FLNA and Sp1 signaling to form an FLNA/GATA4/Sp1 axis to modulate pol III-directed transcription and transformed cell proliferation. Taken together, these results provide novel insights into the regulatory mechanism of pol III-directed transcription.

General transcription factor IIB (TFIIB) and TFIIB-related factor (BRF), are conserved RNA polymerase II/III (RNAPII/III) selectivity factors that are involved in polymerase recruitment and transcription initiation in eukaryotes. Recent findings have shown that plants have evolved a third type of B-factor, plant-specific TFIIB-related protein 1 (pBRP1), which seems to be involved in RNAPI transcription. Here, we extend the repertoire of B-factors in plants by reporting the characterization of a novel TFIIB-related protein, plant-specific TFIIB-related protein 2 (pBRP2), which is found to date only in the Brassicacea family. Unlike other B-factors that are ubiquitously expressed, PBRP2 expression is restricted to reproductive organs and seeds as shown by RT-PCR, immunofluorescence labelling and GUS staining experiments. Interestingly, pbrp2 loss-of-function specifically affects the development of the syncytial endosperm, with both parental contributions required for wild-type development. pBRP2, is the first B-factor to exhibit cell-specific expression and regulation in eukaryotes, and might play a role in enforcing bi-parental reproduction in angiosperms.

Recent X-ray crystallographic studies of Pol II in complex with the general transcription factor (GTF) IIB have begun to provide insights into the mechanism of transcription initiation. These structures have also shed light on the architecture of the transcription preinitiation complex (PIC). However, structural characterization of a functional PIC is still lacking, and even the topological arrangement of the GTFs in the Pol II complex is a matter of contention. We have extended our activity-based affinity crosslinking studies, initially developed to investigate the interaction of bacterial RNA polymerase with σ, to the eukaryotic transcription machinery. Towards that end, we sought to identify GTFs that are within the Pol II active site in a functioning PIC. We provide biochemical evidence that TFIIB is located within ∼9 Å of the -2 site of promoter DNA, where it is positioned to play a role in de novo transcription initiation.

Lung cancer is the leading cause of cancer death worldwide. It has been reported that genetic and epigenetic factors play a crucial role in the onset and evolution of lung cancer. Previous reports have shown that essential transcription factors in embryonic development contribute to this pathology. Runt-related transcription factor (RUNX) proteins belong to a family of master regulators of embryonic developmental programs. Specifically, RUNX2 is the master transcription factor (TF) of osteoblastic differentiation, and it can be involved in pathological conditions such as prostate, thyroid, and lung cancer by regulating apoptosis and mesenchymal-epithelial transition processes. In this paper, we identified TALAM1 (Metastasis Associated Lung Adenocarcinoma Transcript 1) as a genetic target of the RUNX2 TF in lung cancer and then performed functional validation of the main findings.