RESUMO
A long-standing question concerns the role of Z-DNA in transcription. Here we use a deep learning approach DeepZ that predicts Z-flipons based on DNA sequence, structural properties of nucleotides and omics data. We examined Z-flipons that are conserved between human and mouse genomes after generating whole-genome Z-flipon maps and then validated them by orthogonal approaches based on high resolution chemical mapping of Z-DNA and the transformer algorithm Z-DNABERT. For human and mouse, we revealed similar pattern of transcription factors, chromatin remodelers, and histone marks associated with conserved Z-flipons. We found significant enrichment of Z-flipons in alternative and bidirectional promoters associated with neurogenesis genes. We show that conserved Z-flipons are associated with increased experimentally determined transcription reinitiation rates compared to promoters without Z-flipons, but without affecting elongation or pausing. Our findings support a model where Z-flipons engage Transcription Factor E and impact phenotype by enabling the reset of preinitiation complexes when active, and the suppression of gene expression when engaged by repressive chromatin complexes.
Assuntos
DNA , Regiões Promotoras Genéticas , Animais , Humanos , Camundongos , DNA/genética , DNA/metabolismo , Transcrição Gênica , Fatores de Transcrição/metabolismo , Fatores de Transcrição/genética , Montagem e Desmontagem da Cromatina , Iniciação da Transcrição Genética , Cromatina/genética , Cromatina/metabolismo , Aprendizado Profundo , Sequência ConservadaRESUMO
RNA polymerase II (RNAPII) transcription initiates bidirectionally at many human protein-coding genes. Sense transcription usually dominates and leads to messenger RNA production, whereas antisense transcription rapidly terminates. The basis for this directionality is not fully understood. Here, we show that sense transcriptional initiation is more efficient than in the antisense direction, which establishes initial promoter directionality. After transcription begins, the opposing functions of the endonucleolytic subunit of Integrator, INTS11, and cyclin-dependent kinase 9 (CDK9) maintain directionality. Specifically, INTS11 terminates antisense transcription, whereas sense transcription is protected from INTS11-dependent attenuation by CDK9 activity. Strikingly, INTS11 attenuates transcription in both directions upon CDK9 inhibition, and the engineered recruitment of CDK9 desensitises transcription to INTS11. Therefore, the preferential initiation of sense transcription and the opposing activities of CDK9 and INTS11 explain mammalian promoter directionality.
Assuntos
Quinase 9 Dependente de Ciclina , Regiões Promotoras Genéticas , Iniciação da Transcrição Genética , Humanos , Quinase 9 Dependente de Ciclina/metabolismo , Quinase 9 Dependente de Ciclina/genética , Regulação da Expressão Gênica , Proteínas Nucleares , RNA Polimerase II/metabolismo , RNA Polimerase II/genética , Transcrição Gênica , Fatores de Elongação da TranscriçãoRESUMO
Patterns of transcriptional activity are encoded in our genome through regulatory elements such as promoters or enhancers that, paradoxically, contain similar assortments of sequence-specific transcription factor (TF) binding sites1-3. Knowledge of how these sequence motifs encode multiple, often overlapping, gene expression programs is central to understanding gene regulation and how mutations in non-coding DNA manifest in disease4,5. Here, by studying gene regulation from the perspective of individual transcription start sites (TSSs), using natural genetic variation, perturbation of endogenous TF protein levels and massively parallel analysis of natural and synthetic regulatory elements, we show that the effect of TF binding on transcription initiation is position dependent. Analysing TF-binding-site occurrences relative to the TSS, we identified several motifs with highly preferential positioning. We show that these patterns are a combination of a TF's distinct functional profiles-many TFs, including canonical activators such as NRF1, NFY and Sp1, activate or repress transcription initiation depending on their precise position relative to the TSS. As such, TFs and their spacing collectively guide the site and frequency of transcription initiation. More broadly, these findings reveal how similar assortments of TF binding sites can generate distinct gene regulatory outcomes depending on their spatial configuration and how DNA sequence polymorphisms may contribute to transcription variation and disease and underscore a critical role for TSS data in decoding the regulatory information of our genome.
Assuntos
Regulação da Expressão Gênica , Motivos de Nucleotídeos , Regiões Promotoras Genéticas , Fatores de Transcrição , Sítio de Iniciação de Transcrição , Iniciação da Transcrição Genética , Humanos , Sítios de Ligação , Regulação da Expressão Gênica/genética , Genoma Humano/genética , Motivos de Nucleotídeos/genética , Regiões Promotoras Genéticas/genética , Ligação Proteica , Fatores de Transcrição/metabolismo , Variação GenéticaRESUMO
Mitochondrial transcription factor A (TFAM) employs DNA bending to package mitochondrial DNA (mtDNA) into nucleoids and recruit mitochondrial RNA polymerase (POLRMT) at specific promoter sites, light strand promoter (LSP) and heavy strand promoter (HSP). Herein, we characterize the conformational dynamics of TFAM on promoter and non-promoter sequences using single-molecule fluorescence resonance energy transfer (smFRET) and single-molecule protein-induced fluorescence enhancement (smPIFE) methods. The DNA-TFAM complexes dynamically transition between partially and fully bent DNA conformational states. The bending/unbending transition rates and bending stability are DNA sequence-dependent-LSP forms the most stable fully bent complex and the non-specific sequence the least, which correlates with the lifetimes and affinities of TFAM with these DNA sequences. By quantifying the dynamic nature of the DNA-TFAM complexes, our study provides insights into how TFAM acts as a multifunctional protein through the DNA bending states to achieve sequence specificity and fidelity in mitochondrial transcription while performing mtDNA packaging.
Assuntos
Empacotamento do DNA , DNA Mitocondrial , Proteínas de Ligação a DNA , Transferência Ressonante de Energia de Fluorescência , Proteínas Mitocondriais , Conformação de Ácido Nucleico , Regiões Promotoras Genéticas , Fatores de Transcrição , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Proteínas Mitocondriais/metabolismo , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/química , Fatores de Transcrição/metabolismo , Fatores de Transcrição/química , Fatores de Transcrição/genética , DNA Mitocondrial/genética , DNA Mitocondrial/metabolismo , Humanos , Iniciação da Transcrição Genética , Mitocôndrias/metabolismo , Mitocôndrias/genética , Imagem Individual de Molécula , RNA Polimerases Dirigidas por DNA/metabolismo , RNA Polimerases Dirigidas por DNA/química , RNA Polimerases Dirigidas por DNA/genética , Sequência de Bases , Ligação ProteicaRESUMO
Cyclin-dependent kinase 7 (CDK7), part of the general transcription factor TFIIH, promotes gene transcription by phosphorylating the C-terminal domain of RNA polymerase II (RNA Pol II). Here, we combine rapid CDK7 kinase inhibition with multi-omics analysis to unravel the direct functions of CDK7 in human cells. CDK7 inhibition causes RNA Pol II retention at promoters, leading to decreased RNA Pol II initiation and immediate global downregulation of transcript synthesis. Elongation, termination, and recruitment of co-transcriptional factors are not directly affected. Although RNA Pol II, initiation factors, and Mediator accumulate at promoters, RNA Pol II complexes can also proceed into gene bodies without promoter-proximal pausing while retaining initiation factors and Mediator. Further downstream, RNA Pol II phosphorylation increases and initiation factors and Mediator are released, allowing recruitment of elongation factors and an increase in RNA Pol II elongation velocity. Collectively, CDK7 kinase activity promotes the release of initiation factors and Mediator from RNA Pol II, facilitating RNA Pol II escape from the promoter.
Assuntos
Quinase Ativadora de Quinase Dependente de Ciclina , Quinases Ciclina-Dependentes , Regiões Promotoras Genéticas , RNA Polimerase II , Iniciação da Transcrição Genética , Humanos , Quinases Ciclina-Dependentes/metabolismo , Quinases Ciclina-Dependentes/genética , RNA Polimerase II/metabolismo , RNA Polimerase II/genética , Fosforilação , Inibidores de Proteínas Quinases/farmacologia , Complexo Mediador/metabolismo , Complexo Mediador/genética , Células HeLa , Fator de Transcrição TFIIH/metabolismo , Fator de Transcrição TFIIH/genética , Células HEK293RESUMO
Transcription initiation is a vital step in the regulation of eukaryotic gene expression. It can be dysregulated in response to various cellular stressors which is associated with numerous human diseases including cancer. Transcription initiation is facilitated via many gene-specific trans-regulatory elements such as transcription factors, activators, and coactivators through their interactions with transcription pre-initiation complex (PIC). These trans-regulatory elements can uniquely facilitate PIC formation (hence, transcription initiation) in response to cellular nutrient stress. Cellular nutrient stress also regulates the activity of other pathways such as target of rapamycin (TOR) pathway. TOR pathway exhibits distinct regulatory mechanisms of transcriptional activation in response to stress. Like TOR pathway, the cell cycle regulatory pathway is also found to be linked to transcriptional regulation in response to cellular stress. Several transcription factors such as p53, C/EBP Homologous Protein (CHOP), activating transcription factor 6 (ATF6α), E2F, transforming growth factor (TGF)-ß, Adenomatous polyposis coli (APC), SMAD, and MYC have been implicated in regulation of transcription of target genes involved in cell cycle progression, apoptosis, and DNA damage repair pathways. Additionally, cellular metabolic and oxidative stressors have been found to regulate the activity of long non-coding RNAs (lncRNA). LncRNA regulates transcription by upregulating or downregulating the transcription regulatory proteins involved in metabolic and cell signaling pathways. Numerous human diseases, triggered by chronic cellular stressors, are associated with abnormal regulation of transcription. Hence, understanding these mechanisms would help unravel the molecular regulatory insights with potential therapeutic interventions. Therefore, here we emphasize the recent advances of regulation of eukaryotic transcription initiation in response to cellular stress.
Assuntos
Estresse Fisiológico , Humanos , Estresse Fisiológico/genética , Regulação da Expressão Gênica , Iniciação da Transcrição Genética , Animais , Transdução de Sinais , Fatores de Transcrição/metabolismo , Fatores de Transcrição/genética , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Serina-Treonina Quinases TOR/genéticaRESUMO
A deep-learning model reveals the rules that define transcription initiation.
Assuntos
DNA , Sítio de Iniciação de Transcrição , Iniciação da Transcrição Genética , Humanos , Aprendizado Profundo , DNA/genética , Regiões Promotoras GenéticasRESUMO
The transition from transcription initiation to elongation is highly regulated in human cells but remains incompletely understood at the structural level. In particular, it is unclear how interactions between RNA polymerase II (RNA Pol II) and initiation factors are broken to enable promoter escape. Here, we reconstitute RNA Pol II promoter escape in vitro and determine high-resolution structures of initially transcribing complexes containing 8-, 10-, and 12-nt ordered RNAs and two elongation complexes containing 14-nt RNAs. We suggest that promoter escape occurs in three major steps. First, the growing RNA displaces the B-reader element of the initiation factor TFIIB without evicting TFIIB. Second, the rewinding of the transcription bubble coincides with the eviction of TFIIA, TFIIB, and TBP. Third, the binding of DSIF and NELF facilitates TFIIE and TFIIH dissociation, establishing the paused elongation complex. This three-step model for promoter escape fills a gap in our understanding of the initiation-elongation transition of RNA Pol II transcription.
Assuntos
Fosfoproteínas , Regiões Promotoras Genéticas , RNA Polimerase II , Proteína de Ligação a TATA-Box , Fator de Transcrição TFIIB , Fatores de Transcrição , RNA Polimerase II/metabolismo , RNA Polimerase II/genética , Humanos , Fator de Transcrição TFIIB/metabolismo , Fator de Transcrição TFIIB/genética , Proteína de Ligação a TATA-Box/metabolismo , Proteína de Ligação a TATA-Box/genética , Fatores de Transcrição/metabolismo , Fatores de Transcrição/genética , Iniciação da Transcrição Genética , Fator de Transcrição TFIIH/metabolismo , Fator de Transcrição TFIIH/genética , Fator de Transcrição TFIIH/química , Proteínas Nucleares/metabolismo , Proteínas Nucleares/genética , Ligação Proteica , Fator de Transcrição TFIIA/metabolismo , Fator de Transcrição TFIIA/genética , Transcrição Gênica , Elongação da Transcrição Genética , RNA/metabolismo , RNA/genética , Fatores de Transcrição TFII/metabolismo , Fatores de Transcrição TFII/genéticaRESUMO
Transcription initiation is a process that is essential to ensuring the proper function of any gene, yet we still lack a unified understanding of sequence patterns and rules that explain most transcription start sites in the human genome. By predicting transcription initiation at base-pair resolution from sequences with a deep learning-inspired explainable model called Puffin, we show that a small set of simple rules can explain transcription initiation at most human promoters. We identify key sequence patterns that contribute to human promoter activity, each activating transcription with distinct position-specific effects. Furthermore, we explain the sequence basis of bidirectional transcription at promoters, identify the links between promoter sequence and gene expression variation across cell types, and explore the conservation of sequence determinants of transcription initiation across mammalian species.
Assuntos
Genoma Humano , Regiões Promotoras Genéticas , Sítio de Iniciação de Transcrição , Iniciação da Transcrição Genética , Humanos , Aprendizado Profundo , Animais , Sequência de BasesRESUMO
Transcription initiation, the first step in gene expression, has been studied extensively in dilute buffer, a condition which fails to consider the crowded environment in live cells. Recent reports indicate the kinetics of promoter escape is altered in crowded conditions for a consensus bacterial promoter. Here, we use a real-time fluorescence enhancement assay to study the kinetics of unwound bubble formation and promoter escape for three separate promoters. We find that the effect of crowding on transcription initiation is complex, with lower rates of unwound bubble formation, higher rates of promoter escape, and large variations depending on promoter identity. Based on our results, we suggest that altered conditions of crowding inside a live cell can trigger global changes.
Assuntos
Escherichia coli , Regiões Promotoras Genéticas , Iniciação da Transcrição Genética , Escherichia coli/genética , Escherichia coli/metabolismo , Cinética , Substâncias Macromoleculares/metabolismo , Substâncias Macromoleculares/químicaRESUMO
Viruses are master remodelers of the host cell environment in support of infection and virus production. For example, viruses typically regulate cell gene expression through modulating canonical cell promoter activity. Here, we show that Epstein Barr virus (EBV) replication causes 'de novo' transcription initiation at 29674 new transcription start sites throughout the cell genome. De novo transcription initiation is facilitated in part by the unique properties of the viral pre-initiation complex (vPIC) that binds a TATT[T/A]AA, TATA box-like sequence and activates transcription with minimal support by additional transcription factors. Other de novo promoters are driven by the viral transcription factors, Zta and Rta and are influenced by directional proximity to existing canonical cell promoters, a configuration that fosters transcription through existing promoters and transcriptional interference. These studies reveal a new way that viruses interact with the host transcriptome to inhibit host gene expression and they shed light on primal features driving eukaryotic promoter function.
Assuntos
Infecções por Vírus Epstein-Barr , Herpesvirus Humano 4 , Iniciação da Transcrição Genética , Replicação Viral , Humanos , Herpesvirus Humano 4/fisiologia , Interações Hospedeiro-Patógeno , Regiões Promotoras Genéticas , TATA Box , Fatores de Transcrição/metabolismo , Sítio de Iniciação de Transcrição , Transcrição Gênica , Proteínas Virais/metabolismo , Proteínas Virais/genética , Infecções por Vírus Epstein-Barr/metabolismo , Infecções por Vírus Epstein-Barr/virologiaRESUMO
Thirty years have passed since the discovery of the Mediator complex in yeast. We are witnessing breakthroughs and advances that have led to high-resolution structural models of yeast and mammalian Mediators in the preinitiation complex, showing how it is assembled and how it positions the RNA polymerase II and its C-terminal domain (CTD) to facilitate the CTD phosphorylation that initiates transcription. This information may be also used to guide future plant research on the mechanisms of Mediator transcriptional control. Here, we review what we know about the subunit composition and structure of plant Mediators, the roles of the individual subunits and the genetic analyses that pioneered Mediator research, and how transcription factors recruit Mediators to regulatory regions adjoining promoters. What emerges from the research is a Mediator that regulates transcription activity and recruits hormonal signaling modules and histone-modifying activities to set up an off or on transcriptional state that recruits general transcription factors for preinitiation complex assembly.
Assuntos
Complexo Mediador , RNA Polimerase II , RNA Polimerase II/metabolismo , RNA Polimerase II/genética , Complexo Mediador/metabolismo , Complexo Mediador/genética , Complexo Mediador/química , Transcrição Gênica , Fatores de Transcrição/metabolismo , Fatores de Transcrição/genética , Regulação da Expressão Gênica de Plantas , Plantas/genética , Plantas/metabolismo , Plantas/enzimologia , Fosforilação , Iniciação da Transcrição GenéticaRESUMO
Transcription initiation is a complex process, and its mechanism is incompletely understood. We determined the structures of de novo transcribing complexes TC2 to TC17 with RNA polymerase II halted on G-less promoters when nascent RNAs reach 2 to 17 nucleotides in length, respectively. Connecting these structures generated a movie and a working model. As initially synthesized RNA grows, general transcription factors (GTFs) remain bound to the promoter and the transcription bubble expands. Nucleoside triphosphate (NTP)-driven RNA-DNA translocation and template-strand accumulation in a nearly sealed channel may promote the transition from initially transcribing complexes (ITCs) (TC2 to TC9) to early elongation complexes (EECs) (TC10 to TC17). Our study shows dynamic processes of transcription initiation and reveals why ITCs require GTFs and bubble expansion for initial RNA synthesis, whereas EECs need GTF dissociation from the promoter and bubble collapse for promoter escape.
Assuntos
RNA , Fatores Genéricos de Transcrição , Iniciação da Transcrição Genética , RNA Polimerases Dirigidas por DNA/química , RNA/biossíntese , RNA Polimerase II/química , Fatores Genéricos de Transcrição/metabolismo , Humanos , Animais , Sus scrofa , Microscopia Crioeletrônica , Filmes CinematográficosRESUMO
Transcription initiation is a key regulatory step in gene expression during which RNA polymerase (RNAP) initiates RNA synthesis de novo, and the synthesized RNA at a specific length triggers the transition to the elongation phase. Mitochondria recruit a single-subunit RNAP and one or two auxiliary factors to initiate transcription. Previous studies have revealed the molecular architectures of yeast1 and human2 mitochondrial RNAP initiation complexes (ICs). Here we provide a comprehensive, stepwise mechanism of transcription initiation by solving high-resolution cryogenic electron microscopy (cryo-EM) structures of yeast mitochondrial RNAP and the transcription factor Mtf1 catalysing two- to eight-nucleotide RNA synthesis at single-nucleotide addition steps. The growing RNA-DNA is accommodated in the polymerase cleft by template scrunching and non-template reorganization, creating stressed intermediates. During early initiation, non-template strand scrunching and unscrunching destabilize the short two- and three-nucleotide RNAs, triggering abortive synthesis. Subsequently, the non-template reorganizes into a base-stacked staircase-like structure supporting processive five- to eight-nucleotide RNA synthesis. The expanded non-template staircase and highly scrunched template in IC8 destabilize the promoter interactions with Mtf1 to facilitate initiation bubble collapse and promoter escape for the transition from initiation to the elongation complex (EC). The series of transcription initiation steps, each guided by the interplay of multiple structural components, reveal a finely tuned mechanism for potential regulatory control.
Assuntos
Mitocôndrias , Saccharomyces cerevisiae , Iniciação da Transcrição Genética , RNA Polimerases Dirigidas por DNA/metabolismo , RNA Polimerases Dirigidas por DNA/ultraestrutura , Mitocôndrias/enzimologia , Mitocôndrias/genética , Mitocôndrias/ultraestrutura , Nucleotídeos/metabolismo , RNA/biossíntese , RNA/ultraestrutura , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/genética , Microscopia Crioeletrônica , DNA/metabolismo , DNA/ultraestruturaRESUMO
IMPORTANCE: Infection with herpes simplex virus 1 (HSV-1) leads to lifelong infection due to the virus's remarkable ability to control transcription of its own genome, resulting in two transcriptional programs: lytic (highly active) and latent (restricted). The lytic program requires immediate early (IE) proteins to first repress transcription of late viral genes, which then undergo sequential de-repression, leading to a specific sequence of gene expression. Here, we show that the IE ICP4 functions to regulate the cascade by limiting RNA polymerase initiation at immediate early times. However, late viral genes that initiate too early in the absence of ICP4 do not yield mRNA as transcription stalls within gene bodies. It follows that other regulatory steps intercede to prevent elongation of genes at the incorrect time, demonstrating the precise control HSV-1 exerts over its own transcription.
Assuntos
Regulação Viral da Expressão Gênica , Herpesvirus Humano 1 , Proteínas Imediatamente Precoces , Transcrição Gênica , Humanos , Genes Virais/genética , Herpes Simples/virologia , Herpesvirus Humano 1/genética , Proteínas Imediatamente Precoces/deficiência , Proteínas Imediatamente Precoces/metabolismo , Iniciação da Transcrição Genética , Elongação da Transcrição Genética , Terminação da Transcrição GenéticaRESUMO
In addition to being essential for gene expression, transcription is crucial for the maintenance of genome integrity. Here, we undertook a systematic approach, to monitor the assembly kinetics of the pre-initiating RNA Polymerase (Pol) II at promoters at steady state and different stages during recovery from UV irradiation-stress, when pre-initiation and initiation steps have been suggested to be transiently shut down. Taking advantage of the reversible dissociation of pre-initiating Pol II after high salt treatment, we found that de novo recruitment of the available Pol II molecules at active promoters not only persists upon UV at all times tested but occurs significantly faster in the early phase of recovery (2 h) than in unexposed human fibroblasts at the majority of active genes. Our method unveiled groups of genes with significantly different pre-initiation complex (PIC) assembly dynamics after UV that present distinct rates of UV-related mutational signatures in melanoma tumours, providing functional relevance to the importance of keeping transcription initiation active during UV recovery. Our findings uncover novel mechanistic insights further detailing the multilayered transcriptional response to genotoxic stress and link PIC assembly dynamics after exposure to genotoxins with cancer mutational landscapes.
Assuntos
RNA Polimerase II , Iniciação da Transcrição Genética , Humanos , Dano ao DNA , Mutagênese , Regiões Promotoras Genéticas , RNA Polimerase II/genética , RNA Polimerase II/metabolismo , Transcrição Gênica , Raios Ultravioleta , Fibroblastos/metabolismo , Reparo do DNARESUMO
Bacterial transcription initiation requires σ factors for nucleation of the transcription bubble. The canonical housekeeping σ factor, σ70, nucleates DNA melting via recognition of conserved bases of the promoter -10 motif, which are unstacked and captured in pockets of σ70. By contrast, the mechanism of transcription bubble nucleation and formation during the unrelated σN-mediated transcription initiation is poorly understood. Herein, we combine structural and biochemical approaches to establish that σN, like σ70, captures a flipped, unstacked base in a pocket formed between its N-terminal region I (RI) and extra-long helix features. Strikingly, RI inserts into the nascent bubble to stabilize the nucleated bubble prior to engagement of the obligate ATPase activator. Our data suggest a general paradigm of transcription initiation that requires σ factors to nucleate an early melted intermediate prior to productive RNA synthesis.
Assuntos
Escherichia coli , Iniciação da Transcrição Genética , Escherichia coli/química , Escherichia coli/metabolismo , RNA Polimerase Sigma 54/química , Fator sigma/química , Regiões Promotoras Genéticas , Microscopia CrioeletrônicaRESUMO
Most eukaryotic promoter regions are divergently transcribed. As the RNA polymerase II pre-initiation complex (PIC) is intrinsically asymmetric and responsible for transcription in a single direction, it is unknown how divergent transcription arises. Here, the Saccharomyces cerevisiae Mediator complexed with a PIC (Med-PIC) was assembled on a divergent promoter and analyzed by cryoelectron microscopy. The structure reveals two distinct Med-PICs forming a dimer through the Mediator tail module, induced by a homodimeric activator protein localized near the dimerization interface. The tail dimer is associated with â¼80-bp upstream DNA, such that two flanking core promoter regions are positioned and oriented in a suitable form for PIC assembly in opposite directions. Also, cryoelectron tomography visualized the progress of the PIC assembly on the two core promoter regions, providing direct evidence for the role of the Med-PIC dimer in divergent transcription.
Assuntos
RNA Polimerase II , Proteínas de Saccharomyces cerevisiae , RNA Polimerase II/metabolismo , Microscopia Crioeletrônica , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Regiões Promotoras Genéticas , Transcrição Gênica , Complexo Mediador/genética , Iniciação da Transcrição GenéticaRESUMO
Rpc31 is a subunit in the TFIIE-related Rpc82/34/31 heterotrimeric subcomplex of Saccharomyces cerevisiae RNA polymerase III (pol III). Structural analyses of pol III have indicated that the N-terminal region of Rpc31 anchors on Rpc82 and further interacts with the polymerase core and stalk subcomplex. However, structural and functional information for the C-terminal region of Rpc31 is sparse. We conducted a mutational analysis on Rpc31, which uncovered a functional peptide adjacent to the highly conserved Asp-Glu-rich acidic C-terminus. This C-terminal peptide region, termed 'pre-acidic', is important for optimal cell growth, tRNA synthesis, and stable association of Rpc31 in the pre-initiation complex (PIC). Our site-directed photo-cross-linking to map protein interactions within the PIC reveal that this pre-acidic region specifically targets Rpc34 during transcription initiation, but also interacts with the DNA entry surface in free pol III. Thus, we have uncovered a switchable Rpc31 C-terminal region that functions in an initiation-specific protein interaction for pol III transcription.