RESUMO
RNA polymerase II (Pol II) elongation is a critical step in gene expression. Here we found that NDF, which was identified as a bilaterian nucleosome-destabilizing factor, is also a Pol II transcription factor that stimulates elongation with plain DNA templates in the absence of nucleosomes. NDF binds directly to Pol II and enhances elongation by a different mechanism than that used by transcription factor TFIIS. Moreover, yeast Pdp3, which is related to NDF, binds to Pol II and stimulates elongation. Thus, NDF is a Pol II binding transcription elongation factor that is localized over gene bodies and is conserved from yeast to humans.
Assuntos
RNA Polimerase II , Fatores de Transcrição , Humanos , Nucleossomos/metabolismo , RNA Polimerase II/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Transcrição Gênica , Fatores de Elongação da Transcrição/genética , Fatores de Elongação da Transcrição/metabolismoRESUMO
A mechanistic description of metazoan transcription is essential for understanding the molecular processes that govern cellular decisions. To provide structural insights into the DNA recognition step of transcription initiation, we used single-particle electron microscopy (EM) to visualize human TFIID with promoter DNA. This analysis revealed that TFIID coexists in two predominant and distinct structural states that differ by a 100 Å translocation of TFIID's lobe A. The transition between these structural states is modulated by TFIIA, as the presence of TFIIA and promoter DNA facilitates the formation of a rearranged state of TFIID that enables promoter recognition and binding. DNA labeling and footprinting, together with cryo-EM studies, were used to map the locations of TATA, Initiator (Inr), motif ten element (MTE), and downstream core promoter element (DPE) promoter motifs within the TFIID-TFIIA-DNA structure. The existence of two structurally and functionally distinct forms of TFIID suggests that the different conformers may serve as specific targets for the action of regulatory factors.
Assuntos
Regiões Promotoras Genéticas , Fator de Transcrição TFIID/química , Fator de Transcrição TFIID/metabolismo , Transcrição Gênica , Microscopia Crioeletrônica , DNA/genética , Humanos , Conformação Proteica , RNA Polimerase II/química , RNA Polimerase II/metabolismo , TATA Box , Fator de Transcrição TFIIA/metabolismo , Fator de Transcrição TFIID/ultraestrutura , Fatores de Transcrição/química , Fatores de Transcrição/metabolismoRESUMO
Our understanding of transcription by RNA polymerase II (Pol II) is limited by our knowledge of the factors that mediate this critically important process. Here we describe the identification of NDF, a nucleosome-destabilizing factor that facilitates Pol II transcription in chromatin. NDF has a PWWP motif, interacts with nucleosomes near the dyad, destabilizes nucleosomes in an ATP-independent manner, and facilitates transcription by Pol II through nucleosomes in a purified and defined transcription system as well as in cell nuclei. Upon transcriptional induction, NDF is recruited to the transcribed regions of thousands of genes and colocalizes with a subset of H3K36me3-enriched regions. Notably, the recruitment of NDF to gene bodies is accompanied by an increase in the transcript levels of many of the NDF-enriched genes. In addition, the global loss of NDF results in a decrease in the RNA levels of many genes. In humans, NDF is present at high levels in all tested tissue types, is essential in stem cells, and is frequently overexpressed in breast cancer. These findings indicate that NDF is a nucleosome-destabilizing factor that is recruited to gene bodies during transcriptional activation and facilitates Pol II transcription through nucleosomes.
Assuntos
Proteínas de Drosophila/metabolismo , Proteínas Nucleares/metabolismo , Nucleossomos/metabolismo , Oxirredutases/metabolismo , Transcrição Gênica/genética , Motivos de Aminoácidos/genética , Animais , Neoplasias da Mama/genética , Núcleo Celular , Cromatina/metabolismo , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Escherichia coli/genética , Regulação da Expressão Gênica/genética , Histonas/metabolismo , Humanos , Camundongos , Proteínas Nucleares/genética , Oxirredutases/genética , Transporte Proteico , RNA Polimerase II/genética , RNA Polimerase II/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMO
RNA polymerase III (Pol III) transcribes short RNAs required for cell growth. Under stress conditions, the conserved protein Maf1 rapidly represses Pol III transcription. We report the crystal structure of Maf1 and cryo-electron microscopic structures of Pol III, an active Pol III-DNA-RNA complex, and a repressive Pol III-Maf1 complex. Binding of DNA and RNA causes ordering of the Pol III-specific subcomplex C82/34/31 that is required for transcription initiation. Maf1 binds the Pol III clamp and rearranges C82/34/31 at the rim of the active center cleft. This impairs recruitment of Pol III to a complex of promoter DNA with the initiation factors Brf1 and TBP and thus prevents closed complex formation. Maf1 does however not impair binding of a DNA-RNA scaffold and RNA synthesis. These results explain how Maf1 specifically represses transcription initiation from Pol III promoters and indicate that Maf1 also prevents reinitiation by binding Pol III during transcription elongation.
Assuntos
RNA Polimerase III/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/metabolismo , Fatores de Transcrição/metabolismo , Sequência de Aminoácidos , Dados de Sequência Molecular , Complexos Multiproteicos/química , Complexos Multiproteicos/metabolismo , Regiões Promotoras Genéticas , RNA Polimerase III/antagonistas & inibidores , RNA Polimerase III/química , Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Alinhamento de Sequência , Fatores de Transcrição/química , Transcrição GênicaRESUMO
The signals that direct the initiation of transcription ultimately converge at the core promoter, which is the gateway to transcription. Here we provide an overview of the RNA polymerase II core promoter in bilateria (bilaterally symmetric animals). The core promoter is diverse in terms of its composition and function yet is also punctilious, as it acts with strict rules and precision. We additionally describe an expanded view of the core promoter that comprises the classical DNA sequence motifs, sequence-specific DNA-binding transcription factors, chromatin signals, and DNA structure. This model may eventually lead to a more unified conceptual understanding of the core promoter.
Assuntos
Regiões Promotoras Genéticas/genética , RNA Polimerase II/genética , Animais , Cromatina/metabolismo , DNA/química , Motivos de Nucleotídeos/genética , Fatores de Transcrição/genética , Ativação Transcricional/genéticaRESUMO
Chromatin comprises nucleosomes as well as nonnucleosomal histone-DNA particles. Prenucleosomes are rapidly formed histone-DNA particles that can be converted into canonical nucleosomes by a motor protein such as ACF. Here we show that the prenucleosome is a stable conformational isomer of the nucleosome. It consists of a histone octamer associated with â¼ 80 base pair (bp) of DNA, which is located at a position that corresponds to the central 80 bp of a nucleosome core particle. Monomeric prenucleosomes with free flanking DNA do not spontaneously fold into nucleosomes but can be converted into canonical nucleosomes by an ATP-driven motor protein such as ACF or Chd1. In addition, histone H3K56, which is located at the DNA entry and exit points of a canonical nucleosome, is specifically acetylated by p300 in prenucleosomes relative to nucleosomes. Prenucleosomes assembled in vitro exhibit properties that are strikingly similar to those of nonnucleosomal histone-DNA particles in the upstream region of active promoters in vivo. These findings suggest that the prenucleosome, the only known stable conformational isomer of the nucleosome, is related to nonnucleosomal histone-DNA species in the cell.
Assuntos
Cromatina/metabolismo , DNA/metabolismo , Drosophila melanogaster/genética , Nucleossomos/metabolismo , Animais , Drosophila melanogaster/química , Drosophila melanogaster/metabolismo , Histonas/metabolismo , Humanos , Microscopia Eletrônica , Nucleossomos/química , Nucleossomos/genética , Nucleossomos/ultraestrutura , Regiões Promotoras Genéticas , Conformação Proteica , Estabilidade Proteica , Células Sf9RESUMO
CSB/ERCC6 belongs to an orphan subfamily of SWI2/SNF2-related chromatin remodelers and plays crucial roles in gene expression, DNA damage repair, and the maintenance of genome integrity. The molecular basis of chromatin remodeling by Cockayne syndrome B protein (CSB) is not well understood. Here we investigate the molecular mechanism of chromatin remodeling by Rhp26, a Schizosaccharomyces pombe CSB ortholog. The molecular basis of chromatin remodeling and nucleosomal epitope recognition by Rhp26 is distinct from that of canonical chromatin remodelers, such as imitation switch protein (ISWI). We reveal that the remodeling activities are bidirectionally regulated by CSB-specific motifs: the N-terminal leucine-latch motif and the C-terminal coupling motif. Rhp26 remodeling activities depend mainly on H4 tails and to a lesser extent on H3 tails, but not on H2A and H2B tails. Rhp26 promotes the disruption of histone cores and the release of free DNA. Finally, we dissected the distinct contributions of two Rhp26 C-terminal regions to chromatin remodeling and DNA damage repair.
Assuntos
Montagem e Desmontagem da Cromatina , DNA Helicases/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Cromatina/metabolismo , Reparo do DNA , Epitopos , Histonas/metabolismo , SchizosaccharomycesRESUMO
The TCT core promoter element is present in most ribosomal protein (RP) genes in Drosophila and humans. Here we show that TBP (TATA box-binding protein)-related factor TRF2, but not TBP, is required for transcription of the TCT-dependent RP genes. In cells, TCT-dependent transcription, but not TATA-dependent transcription, increases or decreases upon overexpression or depletion of TRF2. In vitro, purified TRF2 activates TCT but not TATA promoters. ChIP-seq (chromatin immunoprecipitation [ChIP] combined with deep sequencing) experiments revealed the preferential localization of TRF2 at TCT versus TATA promoters. Hence, a specialized TRF2-based RNA polymerase II system functions in the synthesis of RPs and complements the RNA polymerase I and III systems.
Assuntos
Drosophila/genética , Drosophila/metabolismo , Proteína 2 de Ligação a Repetições Teloméricas/metabolismo , Transcrição Gênica/genética , Motivos de Aminoácidos , Animais , Linhagem Celular , Expressão Gênica , Regiões Promotoras Genéticas , Transporte Proteico , TATA Box/genética , Proteína de Ligação a TATA-Box/metabolismoRESUMO
The C53 and C37 subunits of RNA polymerase III (pol III) form a subassembly that is required for efficient termination; pol III lacking this subcomplex displays increased processivity of RNA chain elongation. We show that the C53/C37 subcomplex additionally plays a role in formation of the initiation-ready open promoter complex similar to that of the Brf1 N-terminal zinc ribbon domain. In the absence of C53 and C37, the transcription bubble fails to stably propagate to and beyond the transcriptional start site even when the DNA template is supercoiled. The C53/C37 subcomplex also stimulates the formation of an artificially assembled elongation complex from its component DNA and RNA strands. Protein-RNA and protein-DNA photochemical cross-linking analysis places a segment of C53 close to the RNA 3' end and transcribed DNA strand at the catalytic center of the pol III elongation complex. We discuss the implications of these findings for the mechanism of transcriptional termination by pol III and propose a structural as well as functional correspondence between the C53/C37 subcomplex and the RNA polymerase II initiation factor TFIIF.
Assuntos
Regiões Promotoras Genéticas/fisiologia , RNA Polimerase III/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Transcrição Gênica/fisiologia , Domínio Catalítico/fisiologia , Regulação Fúngica da Expressão Gênica/fisiologia , RNA Polimerase II/metabolismo , RNA Polimerase III/genética , RNA Nuclear Pequeno/fisiologia , Proteínas de Saccharomyces cerevisiae/genética , Fatores de Transcrição TFII/metabolismo , Sítio de Iniciação de Transcrição/fisiologiaRESUMO
This article reviews the current state of understanding of the regulated transcription of the bacteriophage T4 late genes, with a focus on the underlying biochemical mechanisms, which turn out to be unique to the T4-related family of phages or significantly different from other bacterial systems. The activator of T4 late transcription is the gene 45 protein (gp45), the sliding clamp of the T4 replisome. Gp45 becomes topologically linked to DNA through the action of its clamp-loader, but it is not site-specifically DNA-bound, as other transcriptional activators are. Gp45 facilitates RNA polymerase recruitment to late promoters by interacting with two phage-encoded polymerase subunits: gp33, the co-activator of T4 late transcription; and gp55, the T4 late promoter recognition protein. The emphasis of this account is on the sites and mechanisms of actions of these three proteins, and on their roles in the formation of transcription-ready open T4 late promoter complexes.
Assuntos
Bacteriófago T4/fisiologia , Genes Virais , Transcrição Gênica , Bacteriófago T4/genética , DNA Viral/metabolismo , Regulação Viral da Expressão Gênica , Regiões Promotoras Genéticas , Ligação Proteica , Proteínas Virais/metabolismoRESUMO
Under growth-limiting conditions, budding yeast shut down transcription of genes of the translation apparatus. Recent studies have shown that this response is signaled, in part, by multiple pathways that converge on Maf1, leading to a change of this protein's phosphorylation state and its relocation to the nucleus, where it represses RNA polymerase III.
Assuntos
Regulação da Expressão Gênica , RNA Polimerase III/genética , Proteínas de Saccharomyces cerevisiae/fisiologia , Saccharomyces cerevisiae/genética , Fatores de Transcrição/fisiologia , Transcrição Gênica/fisiologia , Modelos Genéticos , Fosforilação , RNA Polimerase III/metabolismo , Saccharomyces cerevisiae/crescimento & desenvolvimento , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
Transcription by RNA polymerase II initiates at the core promoter, which is sometimes referred to as the "gateway to transcription." Here, we describe the properties of the RNA polymerase II core promoter in Drosophila The core promoter is at a strategic position in the expression of genes, as it is the site of convergence of the signals that lead to transcriptional activation. Importantly, core promoters are diverse in terms of their structure and function. They are composed of various combinations of sequence motifs such as the TATA box, initiator (Inr), and downstream core promoter element (DPE). Different types of core promoters are transcribed via distinct mechanisms. Moreover, some transcriptional enhancers exhibit specificity for particular types of core promoters. These findings indicate that the core promoter is a central component of the transcriptional apparatus that regulates gene expression.
Assuntos
Drosophila/genética , Regiões Promotoras Genéticas , RNA Polimerase II/metabolismo , Transcrição Gênica , Animais , Drosophila/enzimologia , Proteínas de Drosophila/metabolismo , Ativação TranscricionalRESUMO
Tardigrades, also known as water bears, are animals that can survive extreme conditions. The tardigrade Ramazzottius varieornatus contains a unique nuclear protein termed Dsup, for damage suppressor, which can increase the resistance of human cells to DNA damage under conditions, such as ionizing radiation or hydrogen peroxide treatment, that generate hydroxyl radicals. Here we find that R. varieornatus Dsup is a nucleosome-binding protein that protects chromatin from hydroxyl radicals. Moreover, a Dsup ortholog from the tardigrade Hypsibius exemplaris similarly binds to nucleosomes and protects DNA from hydroxyl radicals. Strikingly, a conserved region in Dsup proteins exhibits sequence similarity to the nucleosome-binding domain of vertebrate HMGN proteins and is functionally important for nucleosome binding and hydroxyl radical protection. These findings suggest that Dsup promotes the survival of tardigrades under diverse conditions by a direct mechanism that involves binding to nucleosomes and protecting chromosomal DNA from hydroxyl radicals.
Assuntos
Dano ao DNA , DNA/metabolismo , Radical Hidroxila/metabolismo , Proteínas Nucleares/metabolismo , Nucleossomos/metabolismo , Tardígrados/enzimologia , AnimaisRESUMO
Transcription of the U6 snRNA gene (SNR6) in Saccharomyces cerevisiae by RNA polymerase III (pol III) requires TFIIIC and its box A and B binding sites. In contrast, TFIIIC has little or no effect on SNR6 transcription with purified components in vitro due to direct recognition of the SNR6 TATA box by TFIIIB. When SNR6 was assembled into chromatin in vitro by use of the Drosophila melanogaster S-190 extract, transcription of these templates with highly purified yeast pol III, TFIIIC, and TFIIIB displayed a near-absolute requirement for TFIIIC but yielded a 5- to 15-fold-higher level of transcription relative to naked DNA (>100-fold activation over repressed chromatin). Analysis of chromatin structure demonstrated that TFIIIC binding leads to remodeling of U6 gene chromatin, resulting in positioning of a nucleosome between boxes A and B. The resulting folding of the intervening DNA into the nucleosome could bring the suboptimally spaced SNR6 box A and B elements into greater proximity and thus facilitate activation of transcription. In the absence of ATP, however, the binding of TFIIIC to box B in chromatin was not accompanied by remodeling and the transcription activation was approximately 35% of that seen in its presence, implying that both TFIIIC binding and ATP-dependent chromatin remodeling were required for the full activation of the gene. Our results suggest that TFIIIC, which is a basal transcription factor of pol III, also plays a direct role in remodeling chromatin on the SNR6 gene.
Assuntos
Cromatina/metabolismo , RNA Nuclear Pequeno/genética , RNA Nuclear Pequeno/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Fatores de Transcrição TFIII/metabolismo , Ativação Transcricional , Trifosfato de Adenosina/metabolismo , Animais , Cromatina/química , Regulação Fúngica da Expressão Gênica , Conformação de Ácido Nucleico , Nucleossomos/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Transcrição GênicaRESUMO
The essential Saccharomyces cerevisiae gene BDP1 encodes a subunit of RNA polymerase III (Pol III) transcription factor (TFIIIB); TATA box binding protein (TBP) and Brf1 are the other subunits of this three-protein complex. Deletion analysis defined three segments of Bdp1 that are essential for viability. A central segment, comprising amino acids 327 to 353, was found to be dispensable, and cells making Bdp1 that was split within this segment, at amino acid 352, are viable. Suppression of bdp1 conditional viability by overexpressing SPT15 and BRF1 identified functional interactions of specific Bdp1 segments with TBP and Brf1, respectively. A Bdp1 deletion near essential segment I was synthetically lethal with overexpression of PCF1-1, a dominant gain-of-function mutation in the second tetracopeptide repeat motif (out of 11) of the Tfc4 (tau(131)) subunit of TFIIIC. The analysis also identifies a connection between Bdp1 and posttranscriptional processing of Pol III transcripts. Yeast genomic library screening identified RPR1 as the specific overexpression suppressor of very slow growth at 37 degrees C due to deletion of Bdp1 amino acids 253 to 269. RPR1 RNA, a Pol III transcript, is the RNA subunit of RNase P, which trims pre-tRNA transcript 5' ends. Maturation of tRNA was found to be aberrant in bdp1-Delta 253-269 cells, and RPR1 transcription with the highly resolved Pol III transcription system in vitro was also diminished when recombinant Bdp1 Delta 253-269 replaced wild-type Bdp1. Physical interaction of RNase P with Bdp1 was demonstrated by coimmunoprecipitation and pull-down assays.
Assuntos
Proteínas de Plantas , Processamento Pós-Transcricional do RNA , RNA de Transferência/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Fatores de Transcrição/metabolismo , Transcrição Gênica , Sequência de Aminoácidos , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Endorribonucleases/metabolismo , Substâncias Macromoleculares , Dados de Sequência Molecular , Fenótipo , Plasmídeos/genética , Plasmídeos/metabolismo , Subunidades Proteicas , RNA Catalítico/metabolismo , RNA Fúngico/metabolismo , Ribonuclease P , Proteínas de Saccharomyces cerevisiae/genética , Alinhamento de Sequência , Fator de Transcrição TFIIIB , Fatores de Transcrição/genéticaRESUMO
The Saccharomyces cerevisiae RPR1 gene encodes the RNA subunit of its RNase P, which processes RNA polymerase (pol) III primary transcripts. RPR1, which is transcribed by pol III, has been isolated as a multicopy suppressor of a specific small internal deletion (amino acids 253-269) in the Bdp1 subunit of transcription factor TFIIIB, the core pol III transcription factor. The selective effect of this Bdp1 deletion on RPR1 transcription has been analyzed in vitro. It is shown that TFIIIC-dependent assembly of TFIIIB on the RPR1 promoter is specifically sensitive to this Bdp1 deletion, leading to gene-specifically defective single-round and multiple-round transcription.
Assuntos
Proteínas de Saccharomyces cerevisiae/genética , Deleção de Sequência , Fator de Transcrição TFIIIB/genética , Sequência de Bases , Dados de Sequência Molecular , Regiões Promotoras Genéticas , Proteínas de Saccharomyces cerevisiae/biossíntese , Fator de Transcrição TFIIIB/biossíntese , Fatores de Transcrição TFIII/fisiologia , Transcrição Gênica/genéticaRESUMO
HARP (SMARCAL1, MARCAL1) is an annealing helicase that functions in the repair and restart of damaged DNA replication forks through its DNA branch migration and replication fork regression activities. HARP is conserved among metazoans. HARP from invertebrates differs by the absence of one of the two HARP-specific domain repeats found in vertebrates. The annealing helicase and branch migration activity of invertebrate HARP has not been documented. We found that HARP from Drosophila melanogaster retains the annealing helicase activity of human HARP, the ability to disrupt D-loops and to branch migrate Holliday junctions, but fails to regress model DNA replication fork structures. A comparison of human and Drosophila HARP on additional substrates revealed that both HARPs are competent in branch migrating a bidirectional replication bubble composed of either DNA:DNA or RNA:DNA hybrid. Human, but not Drosophila, HARP is also capable of regressing a replication fork structure containing a highly stable poly rG:dC hybrid. Persistent RNA:DNA hybrids in vivo can lead to replication fork arrest and genome instability. The ability of HARP to strand transfer hybrids may signify a hybrid removal function for this enzyme, in vivo.
Assuntos
DNA Helicases/metabolismo , Replicação do DNA , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/enzimologia , Drosophila melanogaster/genética , Animais , DNA Helicases/genética , DNA Cruciforme , Proteínas de Drosophila/genética , Instabilidade Genômica , Humanos , Ligação ProteicaRESUMO
Nielsen et al. (Reports, 28 June 2013, p. 1577) characterized their RNA polymerase III (Pol III) preparation and concluded that it requires an RNA hairpin/duplex structure for terminating transcription. We could not corroborate their findings using bona fide Pol III from two laboratory sources. We show that Pol III efficiently terminates transcription in the absence of a hairpin/duplex in vitro and in vivo.
Assuntos
RNA Polimerase III/metabolismo , Saccharomyces cerevisiae/enzimologia , Terminação da Transcrição GenéticaRESUMO
A major limitation of chromatin immunoprecipitation lies in the challenge of measuring the immunoprecipitation effectiveness of different proteins and antibodies and the resultant inability to compare the occupancies of different DNA-binding proteins. Here we present the implementation of a quantitative chromatin immunoprecipitation assay in the RNA polymerase III (pol III) system that allowed us to measure the absolute in vivo occupancy of pol III and its two transcription factors, TFIIIC and TFIIIB, on a subset of pol III genes. The crucial point of our analysis was devising a method that allows the accurate determination of the immunoprecipitation efficiency for each protein. We achieved this by spiking every immunoprecipitation reaction with the formaldehyde cross-linked in vitro counterparts of TFIIIB-, TFIIIC-, and pol III-DNA complexes, measuring the in vitro occupancies of the corresponding factors on a DNA probe and determining probe recovery by quantitative PCR. Analysis of nine pol III-transcribed genes with diverse sequence characteristics showed a very high occupancy by TFIIIB and pol III (pol III occupancy being generally approximately 70% of TFIIIB occupancy) and a TFIIIC occupancy that ranged between approximately 5 and 25%. Current data suggest that TFIIIC is released during transcription in vitro, and it has been proposed that TFIIIB suffices for pol III recruitment in vivo. Our findings point to the transient nature of the TFIIIC-DNA interaction in vivo, with no significant counter-correlation between pol III and TFIIIC occupancy and instead to a dependence of TFIIIB-DNA and TFIIIC-DNA complex maintenance in vivo on pol III function.
Assuntos
RNA Polimerase III/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Fator de Transcrição TFIIIB/metabolismo , Fatores de Transcrição TFIII/metabolismo , Transcrição Gênica/fisiologia , DNA Fúngico/metabolismoRESUMO
The binding of the RNA polymerase III (pol III) transcription factor TFIIIC to the box A intragenic promoter element of tRNA genes specifies the placement of TFIIIB on upstream-lying DNA. In turn, TFIIIB recruits pol III to the promoter and specifies transcription initiating 17-19 base pairs upstream of box A. The resolution of the pol III transcription apparatus into recombinant TFIIIB, highly purified TFIIIC, and pol III is accompanied by a loss of precision in specifying where transcription initiation occurs due to heterogeneous placement of TFIIIB. In this paper we show that Nhp6a, an abundant high mobility group B (HMGB) family, non-sequence-specific DNA-binding protein in Saccharomyces cerevisiae restores transcriptional initiation fidelity to this highly purified in vitro system. Restoration of initiation fidelity requires the presence of Nhp6a prior to TFIIIB-DNA complex formation. Chemical nuclease footprinting of TFIIIC- and TFIIIB-TFIIIC-DNA complexes reveals that Nhp6a markedly alters the TFIIIC footprint over box A and reduces the size of the TFIIIB footprint on upstream DNA sequence. Analyses of unprocessed tRNAs from yeast lacking Nhp6a and its closely related paralogue Nhp6b demonstrate that Nhp6 is required for transcriptional initiation fidelity of some but not all tRNA genes, in vivo.