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1.
Genes Dev ; 32(17-18): 1252-1265, 2018 09 01.
Artículo en Inglés | MEDLINE | ID: mdl-30108132

RESUMEN

The transcriptional coactivators Mediator and two histone acetyltransferase (HAT) complexes, NuA4 and SAGA, play global roles in transcriptional activation. Here we explore the relative contributions of these factors to RNA polymerase II association at specific genes and gene classes by rapid nuclear depletion of key complex subunits. We show that the NuA4 HAT Esa1 differentially affects certain groups of genes, whereas the SAGA HAT Gcn5 has a weaker but more uniform effect. Relative dependence on Esa1 and Tra1, a shared component of NuA4 and SAGA, distinguishes two large groups of coregulated growth-promoting genes. In contrast, we show that the activity of Mediator is particularly important at a separate, small set of highly transcribed TATA-box-containing genes. Our analysis indicates that at least three distinct combinations of coactivator deployment are used to generate moderate or high transcription levels and suggests that each may be associated with distinct forms of regulation.


Asunto(s)
Regulación Fúngica de la Expresión Génica , Histona Acetiltransferasas/fisiología , Complejo Mediador/fisiología , Proteínas de Saccharomyces cerevisiae/fisiología , Saccharomyces cerevisiae/genética , Activación Transcripcional , Acetilación , Histonas/metabolismo , Complejo Mediador/metabolismo , Estrés Oxidativo/genética , Regiones Promotoras Genéticas , Saccharomyces cerevisiae/enzimología , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteína de Unión a TATA-Box/metabolismo , Transcripción Genética
2.
Mol Cell ; 68(1): 130-143.e5, 2017 Oct 05.
Artículo en Inglés | MEDLINE | ID: mdl-28918903

RESUMEN

Prior studies suggested that SAGA and TFIID are alternative factors that promote RNA polymerase II transcription, with about 10% of genes in S. cerevisiae dependent on SAGA. We reassessed the role of SAGA by mapping its genome-wide location and role in global transcription in budding yeast. We find that SAGA maps to the UAS elements of most genes, overlapping with Mediator binding and irrespective of previous designations of SAGA- or TFIID-dominated genes. Disruption of SAGA through mutation or rapid subunit depletion reduces transcription from nearly all genes, measured by newly synthesized RNA. We also find that the acetyltransferase Gcn5 synergizes with Spt3 to promote global transcription and that Spt3 functions to stimulate TBP recruitment at all tested genes. Our data demonstrate that SAGA acts as a general cofactor required for essentially all RNA polymerase II transcription and is not consistent with the previous classification of SAGA- and TFIID-dominated genes.


Asunto(s)
Regulación Fúngica de la Expresión Génica , Histona Acetiltransferasas/genética , ARN Polimerasa II/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Proteína de Unión a TATA-Box/genética , Transactivadores/genética , Factores de Transcripción/genética , Eliminación de Gen , Histona Acetiltransferasas/metabolismo , Regiones Promotoras Genéticas , Subunidades de Proteína/genética , Subunidades de Proteína/metabolismo , ARN Polimerasa II/metabolismo , ARN de Hongos/genética , ARN de Hongos/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Factores Asociados con la Proteína de Unión a TATA/genética , Factores Asociados con la Proteína de Unión a TATA/metabolismo , Proteína de Unión a TATA-Box/metabolismo , Transactivadores/metabolismo , Factores de Transcripción/metabolismo , Transcripción Genética
3.
Nucleic Acids Res ; 50(18): e106, 2022 10 14.
Artículo en Inglés | MEDLINE | ID: mdl-35871301

RESUMEN

With the rapid growth of synthetic messenger RNA (mRNA)-based therapeutics and vaccines, the development of analytical tools for characterization of long, complex RNAs has become essential. Tandem liquid chromatography-mass spectrometry (LC-MS/MS) permits direct assessment of the mRNA primary sequence and modifications thereof without conversion to cDNA or amplification. It relies upon digestion of mRNA with site-specific endoribonucleases to generate pools of short oligonucleotides that are then amenable to MS-based sequence analysis. Here, we showed that the uridine-specific human endoribonuclease hRNase 4 improves mRNA sequence coverage, in comparison with the benchmark enzyme RNase T1, by producing a larger population of uniquely mappable cleavage products. We deployed hRNase 4 to characterize mRNAs fully substituted with 1-methylpseudouridine (m1Ψ) or 5-methoxyuridine (mo5U), as well as mRNAs selectively depleted of uridine-two key strategies to reduce synthetic mRNA immunogenicity. Lastly, we demonstrated that hRNase 4 enables direct assessment of the 5' cap incorporation into in vitro transcribed mRNA. Collectively, this study highlights the power of hRNase 4 to interrogate mRNA sequence, identity, and modifications by LC-MS/MS.


Asunto(s)
Endorribonucleasas/química , ARN Mensajero/química , Análisis de Secuencia de ARN/métodos , Espectrometría de Masas en Tándem , Cromatografía Liquida/métodos , ADN Complementario , Humanos , Oligonucleótidos/análisis , ARN Mensajero/genética , Ribonucleasa T1/metabolismo , Espectrometría de Masas en Tándem/métodos
4.
Nucleic Acids Res ; 49(9): 5265-5277, 2021 05 21.
Artículo en Inglés | MEDLINE | ID: mdl-33885787

RESUMEN

Since its initial characterization, Escherichia coli RNase I has been described as a single-strand specific RNA endonuclease that cleaves its substrate in a largely sequence independent manner. Here, we describe a strong calcium (Ca2+)-dependent activity of RNase I on double-stranded RNA (dsRNA), and a Ca2+-dependent novel hybridase activity, digesting the RNA strand in a DNA:RNA hybrid. Surprisingly, Ca2+ does not affect the activity of RNase I on single stranded RNA (ssRNA), suggesting a specific role for Ca2+ in the modulation of RNase I activity. Mutation of a previously overlooked Ca2+ binding site on RNase I resulted in a gain-of-function enzyme that is highly active on dsRNA and could no longer be stimulated by the metal. In summary, our data imply that native RNase I contains a bound Ca2+, allowing it to target both single- and double-stranded RNAs, thus having a broader substrate specificity than originally proposed for this traditional enzyme. In addition, the finding that the dsRNase activity, and not the ssRNase activity, is associated with the Ca2+-dependency of RNase I may be useful as a tool in applied molecular biology.


Asunto(s)
Calcio/metabolismo , Endorribonucleasas/metabolismo , ARN Bicatenario/metabolismo , Sustitución de Aminoácidos , ADN , Endorribonucleasas/química , Endorribonucleasas/genética , Metales/metabolismo , ARN/metabolismo , Ribonucleasas/metabolismo , Especificidad por Sustrato
5.
Protein Expr Purif ; 190: 105987, 2022 02.
Artículo en Inglés | MEDLINE | ID: mdl-34637916

RESUMEN

Combinations of ribonucleases (RNases) are commonly used to digest RNA into oligoribonucleotide fragments prior to liquid chromatography-mass spectrometry (LC-MS) analysis. The distribution of the RNase target sequences or nucleobase sites within an RNA molecule is critical for achieving a high mapping coverage. Cusativin and MC1 are nucleotide-specific endoribonucleases encoded in the cucumber and bitter melon genomes, respectively. Their high specificity for cytidine (Cusativin) and uridine (MC1) make them ideal molecular biology tools for RNA modification mapping. However, heterogenous recombinant expression of either enzyme has been challenging because of their high toxicity to expression hosts and the requirement of posttranslational modifications. Here, we present two highly efficient and time-saving protocols that overcome these hurdles and enhance the expression and purification of these RNases. We first purified MC1 and Cusativin from bacteria by expressing and shuttling both enzymes to the periplasm as MBP-fusion proteins in T7 Express lysY/IqE. coli strain at low temperature. The RNases were enriched using amylose affinity chromatography, followed by a subsequent purification via a C-terminal 6xHIS tag. This fast, two-step purification allows for the purification of highly active recombinant RNases significantly surpassing yields reported in previous studies. In addition, we expressed and purified a Cusativin-CBD fusion enzyme in P. pastoris using chitin magnetic beads. Both Cusativin variants exhibited a similar sequence preference, suggesting that neither posttranslational modifications nor the epitope-tags have a substantial effect on the sequence specificity of the enzyme.


Asunto(s)
Endorribonucleasas , Escherichia coli , Expresión Génica , Ribonucleasas , Endorribonucleasas/biosíntesis , Endorribonucleasas/química , Endorribonucleasas/genética , Endorribonucleasas/aislamiento & purificación , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Ribonucleasas/biosíntesis , Ribonucleasas/química , Ribonucleasas/genética , Ribonucleasas/aislamiento & purificación
7.
EMBO J ; 35(22): 2435-2446, 2016 11 15.
Artículo en Inglés | MEDLINE | ID: mdl-27797823

RESUMEN

Mediator is a conserved, essential transcriptional coactivator complex, but its in vivo functions have remained unclear due to conflicting data regarding its genome-wide binding pattern obtained by genome-wide ChIP Here, we used ChEC-seq, a method orthogonal to ChIP, to generate a high-resolution map of Mediator binding to the yeast genome. We find that Mediator associates with upstream activating sequences (UASs) rather than the core promoter or gene body under all conditions tested. Mediator occupancy is surprisingly correlated with transcription levels at only a small fraction of genes. Using the same approach to map TFIID, we find that TFIID is associated with both TFIID- and SAGA-dependent genes and that TFIID and Mediator occupancy is cooperative. Our results clarify Mediator recruitment and binding to the genome, showing that Mediator binding to UASs is widespread, partially uncoupled from transcription, and mediated in part by TFIID.


Asunto(s)
ADN de Hongos/metabolismo , Complejo Mediador/metabolismo , Secuencias Reguladoras de Ácidos Nucleicos , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteína de Unión a TATA-Box/metabolismo , Transcripción Genética , Inmunoprecipitación de Cromatina , Unión Proteica
8.
Trends Biochem Sci ; 38(12): 603-11, 2013 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-24120742

RESUMEN

Transcriptional regulation is one of the most important steps in control of cell identity, growth, differentiation, and development. Many signaling pathways controlling these processes ultimately target the core transcription machinery that, for protein coding genes, consists of RNA polymerase II (Pol II) and the general transcription factors (GTFs). New studies on the structure and mechanism of the core assembly and how it interfaces with promoter DNA and coactivator complexes have given tremendous insight into early steps in the initiation process, genome-wide binding, and mechanisms conserved for all nuclear and archaeal Pols. Here, we review recent developments in dissecting the architecture of the Pol II core machinery with a focus on early and regulated steps in transcription initiation.


Asunto(s)
ARN Polimerasa II/metabolismo , Transcripción Genética , Modelos Moleculares , ARN Polimerasa II/química
9.
PLoS One ; 18(11): e0291267, 2023.
Artículo en Inglés | MEDLINE | ID: mdl-37939088

RESUMEN

The chemical modification of RNA bases represents a ubiquitous activity that spans all domains of life. Pseudouridylation is the most common RNA modification and is observed within tRNA, rRNA, ncRNA and mRNAs. Pseudouridine synthase or 'PUS' enzymes include those that rely on guide RNA molecules and others that function as 'stand-alone' enzymes. Among the latter, several have been shown to modify mRNA transcripts. Although recent studies have defined the structural requirements for RNA to act as a PUS target, the mechanisms by which PUS1 recognizes these target sequences in mRNA are not well understood. Here we describe the crystal structure of yeast PUS1 bound to an RNA target that we identified as being a hot spot for PUS1-interaction within a model mRNA at 2.4 Å resolution. The enzyme recognizes and binds both strands in a helical RNA duplex, and thus guides the RNA containing the target uridine to the active site for subsequent modification of the transcript. The study also allows us to show the divergence of related PUS1 enzymes and their corresponding RNA target specificities, and to speculate on the basis by which PUS1 binds and modifies mRNA or tRNA substrates.


Asunto(s)
Transferasas Intramoleculares , Saccharomyces cerevisiae , Saccharomyces cerevisiae/metabolismo , ARN Mensajero/metabolismo , ARN/metabolismo , Transferasas Intramoleculares/genética , Transferasas Intramoleculares/metabolismo , ARN de Transferencia/metabolismo , Seudouridina/metabolismo
10.
Nucleic Acids Res ; 38(6): 1950-63, 2010 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-20040576

RESUMEN

The lower jaws of archaeal RNA polymerase and eukaryotic RNA polymerase II include orthologous subunits H and Rpb5, respectively. The tertiary structure of H is very similar to the structure of the C-terminal domain of Rpb5, and both subunits are proximal to downstream DNA in pre-initiation complexes. Analyses of reconstituted euryarchaeal polymerase lacking subunit H revealed that H is important for open complex formation and initial transcription. Eukaryotic Rpb5 rescues activity of the DeltaH enzyme indicating a strong conservation of function for this subunit from archaea to eukaryotes. Photochemical cross-linking in elongation complexes revealed a striking structural rearrangement of RNA polymerase, bringing subunit H near the transcribed DNA strand one helical turn downstream of the active center, in contrast to the positioning observed in preinitiation complexes. The rearrangement of subunits H and A'' suggest a major conformational change in the archaeal RNAP lower jaw upon formation of the elongation complex.


Asunto(s)
Proteínas Arqueales/química , ARN Polimerasas Dirigidas por ADN/química , Subunidades de Proteína/química , Transcripción Genética , Proteínas Arqueales/metabolismo , Secuencia de Bases , ADN/química , ADN/metabolismo , ARN Polimerasas Dirigidas por ADN/metabolismo , Modelos Moleculares , Datos de Secuencia Molecular , Regiones Promotoras Genéticas , Subunidades de Proteína/metabolismo , ARN Polimerasa II/química , ARN Polimerasa II/metabolismo
11.
Biochem Soc Trans ; 37(Pt 1): 18-22, 2009 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-19143595

RESUMEN

The recent success in reconstitution of RNAPs (RNA polymerases) from hyperthermophilic archaea from bacterially expressed purified subunits opens the way for detailed structure-function analyses of multisubunit RNAPs. The archaeal enzyme shows close structural similarity to eukaryotic RNAP, particularly to polymerase II, and can therefore be used as model for analyses of the eukaryotic transcriptional machinery. The cleft loops in the active centre of RNAP were deleted and modified to unravel their function in interaction with nucleic acids during transcription. The rudder, lid and fork 2 cleft loops were required for promoter-directed initiation and elongation, the rudder was essential for open complex formation. Analyses of transcripts from heteroduplex templates containing stable open complexes revealed that bubble reclosure is required for RNA displacement during elongation. Archaeal transcription systems contain, besides the orthologues of the eukaryotic transcription factors TBP (TATA-box-binding protein) and TF (transcription factor) IIB, an orthologue of the N-terminal part of the alpha subunit of eukaryotic TFIIE, called TFE, whose function is poorly understood. Recent analyses revealed that TFE is involved in open complex formation and, in striking contrast with eukaryotic TFIIE, is also present in elongation complexes. Recombinant archaeal RNAPs lacking specific subunits were used to investigate the functions of smaller subunits. These studies revealed that the subunits P and H, the orthologues of eukaryotic Rpb12 and Rpb5, were not required for RNAP assembly. Subunit P was essential for open complex formation, and the DeltaH enzyme was greatly impaired in all assays, with the exception of promoter recruitment. Recent reconstitution studies indicate that Rpb12 and Rpb5 can be incorporated into archaeal RNAP and can complement for the function of the corresponding archaeal subunit in in vitro transcription assays.


Asunto(s)
Archaea/enzimología , Proteínas Arqueales/genética , ARN Polimerasas Dirigidas por ADN/genética , Proteínas Recombinantes/metabolismo , Secuencia de Aminoácidos , Proteínas Arqueales/química , Análisis Mutacional de ADN , ARN Polimerasas Dirigidas por ADN/química , Células Eucariotas/enzimología , Datos de Secuencia Molecular , Subunidades de Proteína/metabolismo , Proteínas Recombinantes/química , Factores de Transcripción TFII/metabolismo , Transcripción Genética
12.
J Bacteriol ; 190(1): 157-67, 2008 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-17965161

RESUMEN

The genome of the hyperthermophile archaeon Pyrococcus furiosus encodes two transcription factor B (TFB) paralogs, one of which (TFB1) was previously characterized in transcription initiation. The second TFB (TFB2) is unusual in that it lacks recognizable homology to the archaeal TFB/eukaryotic TFIIB B-finger motif. TFB2 functions poorly in promoter-dependent transcription initiation, but photochemical cross-linking experiments indicated that the orientation and occupancy of transcription complexes formed with TFB2 at the strong gdh promoter are similar to the orientation and occupancy of transcription complexes formed with TFB1. Initiation complexes formed by TFB2 display a promoter opening defect that can be bypassed with a preformed transcription bubble, suggesting a mechanism to explain the low TFB2 transcription activity. Domain swaps between TFB1 and TFB2 showed that the low activity of TFB2 is determined mainly by its N terminus. The low activity of TFB2 in promoter opening and transcription can be partially relieved by transcription factor E (TFE). The results indicate that the TFB N-terminal region, containing conserved Zn ribbon and B-finger motifs, is important in promoter opening and that TFE can compensate for defects in the N terminus through enhancement of promoter opening.


Asunto(s)
Proteínas Arqueales/genética , Pyrococcus furiosus/genética , Factor de Transcripción TFIIB/genética , Transcripción Genética , Secuencia Conservada , ADN de Archaea/genética , ADN de Archaea/aislamiento & purificación , Genoma Arqueal , Iniciación de la Cadena Peptídica Traduccional , Regiones Promotoras Genéticas
13.
Science ; 362(6421)2018 12 21.
Artículo en Inglés | MEDLINE | ID: mdl-30442764

RESUMEN

The general transcription factor IID (TFIID) is a critical component of the eukaryotic transcription preinitiation complex (PIC) and is responsible for recognizing the core promoter DNA and initiating PIC assembly. We used cryo-electron microscopy, chemical cross-linking mass spectrometry, and biochemical reconstitution to determine the complete molecular architecture of TFIID and define the conformational landscape of TFIID in the process of TATA box-binding protein (TBP) loading onto promoter DNA. Our structural analysis revealed five structural states of TFIID in the presence of TFIIA and promoter DNA, showing that the initial binding of TFIID to the downstream promoter positions the upstream DNA and facilitates scanning of TBP for a TATA box and the subsequent engagement of the promoter. Our findings provide a mechanistic model for the specific loading of TBP by TFIID onto the promoter.


Asunto(s)
Regiones Promotoras Genéticas , Proteína de Unión a TATA-Box/química , Factor de Transcripción TFIID/química , Iniciación de la Transcripción Genética , Reactivos de Enlaces Cruzados/química , Microscopía por Crioelectrón , ADN/química , ADN/metabolismo , Humanos , Unión Proteica , Dominios Proteicos , Multimerización de Proteína , Estabilidad Proteica
14.
Transcription ; 8(3): 169-174, 2017 05 27.
Artículo en Inglés | MEDLINE | ID: mdl-28301289

RESUMEN

Mediator is a conserved and essential coactivator complex broadly required for RNA polymerase II (RNAPII) transcription. Recent genome-wide studies of Mediator binding in budding yeast have revealed new insights into the functions of this critical complex and raised new questions about its role in the regulation of gene expression.


Asunto(s)
Regulación Fúngica de la Expresión Génica/fisiología , Genoma Fúngico/fisiología , Complejo Mediador , ARN Polimerasa II , Proteínas de Schizosaccharomyces pombe , Schizosaccharomyces , Complejo Mediador/genética , Complejo Mediador/metabolismo , ARN Polimerasa II/genética , ARN Polimerasa II/metabolismo , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismo , Proteínas de Schizosaccharomyces pombe/genética , Proteínas de Schizosaccharomyces pombe/metabolismo
15.
J Vis Exp ; (124)2017 06 03.
Artículo en Inglés | MEDLINE | ID: mdl-28605389

RESUMEN

Genome-wide mapping of protein-DNA interactions is critical for understanding gene regulation, chromatin remodeling, and other chromatin-resident processes. Formaldehyde crosslinking followed by chromatin immunoprecipitation and high-throughput sequencing (X-ChIP-seq) has been used to gain many valuable insights into genome biology. However, X-ChIP-seq has notable limitations linked to crosslinking and sonication. Native ChIP avoids these drawbacks by omitting crosslinking, but often results in poor recovery of chromatin-bound proteins. In addition, all ChIP-based methods are subject to antibody quality considerations. Enzymatic methods for mapping protein-DNA interactions, which involve fusion of a protein of interest to a DNA-modifying enzyme, have also been used to map protein-DNA interactions. We recently combined one such method, chromatin endogenous cleavage (ChEC), with high-throughput sequencing as ChEC-seq. ChEC-seq relies on fusion of a chromatin-associated protein of interest to micrococcal nuclease (MNase) to generate targeted DNA cleavage in the presence of calcium in living cells. ChEC-seq is not based on immunoprecipitation and so circumvents potential concerns with crosslinking, sonication, chromatin solubilization, and antibody quality while providing high resolution mapping with minimal background signal. We envision that ChEC-seq will be a powerful counterpart to ChIP, providing an independent means by which to both validate ChIP-seq findings and discover new insights into genomic regulation.


Asunto(s)
Inmunoprecipitación de Cromatina/métodos , Mapeo Cromosómico , Proteínas de Unión al ADN/metabolismo , ADN/metabolismo , Secuenciación de Nucleótidos de Alto Rendimiento , Saccharomyces cerevisiae/genética , Nucleasa Microcócica/metabolismo , Mapeo de Interacción de Proteínas
16.
Nat Struct Mol Biol ; 19(8): 788-96, 2012 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-22751016

RESUMEN

Yeast RNA polymerase II (Pol II) general transcription factor TFIIE and the TFIIH subunit Ssl2 (yeast ortholog of mammalian XPB) function in the transition of the preinitiation complex (PIC) to the open complex. We show that the three TFIIE winged-helix (WH) domains form a heterodimer, with the Tfa1 (TFIIEα) WH binding the Pol II clamp and the Tfa2 (TFIIEß) tandem WH domain encircling promoter DNA that becomes single-stranded in the open complex. Ssl2 lies adjacent to TFIIE, enclosing downstream promoter DNA. Unlike previous proposals, comparison of the PIC and open-complex models strongly suggests that Ssl2 promotes DNA opening by functioning as a double-stranded-DNA translocase, feeding 15 base pairs into the Pol II cleft. Right-handed threading of DNA through the Ssl2 binding groove, combined with the fixed position of upstream promoter DNA, leads to DNA unwinding and the open state.


Asunto(s)
ARN Polimerasa II/química , ARN Polimerasa II/metabolismo , Factores de Transcripción TFII/química , Factores de Transcripción TFII/metabolismo , Adenosina Trifosfato/metabolismo , Sitios de Unión/genética , ADN Helicasas/química , ADN Helicasas/genética , ADN Helicasas/metabolismo , ADN de Hongos/química , ADN de Hongos/genética , ADN de Hongos/metabolismo , Dimerización , Modelos Moleculares , Complejos Multiproteicos/química , Complejos Multiproteicos/metabolismo , Mutagénesis Sitio-Dirigida , Conformación de Ácido Nucleico , Desnaturalización de Ácido Nucleico , Regiones Promotoras Genéticas , Dominios y Motivos de Interacción de Proteínas , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Factor de Transcripción TFIIH/química , Factor de Transcripción TFIIH/genética , Factor de Transcripción TFIIH/metabolismo , Factores de Transcripción TFII/genética
17.
J Biol Chem ; 282(15): 11047-57, 2007 Apr 13.
Artículo en Inglés | MEDLINE | ID: mdl-17311916

RESUMEN

RNA polymerases from Archaea and Eukaryotes consist of a core enzyme associated with a dimeric E'F (Rpb7/Rpb4) subcomplex but the functional contribution of the two subunit subcomplexes to the transcription process is poorly understood. Here we report the reconstitution of the 11-subunit RNA polymerase and of the core enzyme from the hyperthermophilic Archaeon Pyrococcus furiosus. The core enzyme showed significant activity between 70 and 80 degrees C but was almost inactive at 60 degrees C. E' stimulated the activity of the core enzyme at 60 degrees C, dramatically suggesting an important role of this subunit at low growth temperatures. Subunit F did not contribute significantly to catalytic activity. Permanganate footprinting at low temperatures dissected the contributions of the core enzyme, subunit E', and of archaeal TFE to open complex formation. Opening in the -2 and -4 region could be achieved by the core enzyme, subunit E' stimulated bubble formation in general and opening at the upstream end of the transcription bubble was preferably stimulated by TFE. Analyses of the kinetic stabilities of open complexes revealed an unexpected E'-independent role of TFE in the stabilization of open complexes.


Asunto(s)
Proteínas Arqueales/metabolismo , ARN Polimerasa II/metabolismo , Transcripción Genética/genética , Proteínas Arqueales/genética , Proteínas Arqueales/aislamiento & purificación , Regiones Promotoras Genéticas/genética , Unión Proteica , Subunidades de Proteína/genética , Subunidades de Proteína/aislamiento & purificación , Subunidades de Proteína/metabolismo , Pyrococcus furiosus/enzimología , Pyrococcus furiosus/genética , ARN Polimerasa II/genética , ARN Polimerasa II/aislamiento & purificación , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Temperatura
18.
J Biol Chem ; 282(49): 35482-90, 2007 Dec 07.
Artículo en Inglés | MEDLINE | ID: mdl-17921145

RESUMEN

A homologue of the N-terminal domain of the alpha subunit of the general eukaryotic transcription factor TFE is encoded in the genomes of all sequenced archaea, but the position of archaeal TFE in transcription complexes has not yet been defined. We show here that TFE binds nonspecifically to single-stranded DNA, and photochemical cross-linking revealed TFE binding to a preformed open transcription bubble. In preinitiation complexes, the N-terminal part of TFE containing a winged helix-turn-helix motif is cross-linked specifically to DNA of the nontemplate DNA strand at positions -9 and -11. In complexes stalled at +20, TFE cross-linked specifically to positions +9, +11, and +16 of the non-template strand. Analyses of transcription complexes stalled at position +20 revealed a TFE-dependent increase of the resumption efficiency of stalled RNA polymerase and a TFE-induced enhanced permanganate sensitivity of thymine residues in the transcription bubble. These results demonstrate the presence of TFE in early elongation complexes and suggest a role of TFE in stabilization of the transcription bubble during elongation.


Asunto(s)
Proteínas Arqueales/metabolismo , ARN Polimerasas Dirigidas por ADN/metabolismo , Factores de Transcripción/metabolismo , Transcripción Genética/fisiología , Secuencias de Aminoácidos/fisiología , Proteínas Arqueales/química , Sistema Libre de Células/química , Sistema Libre de Células/metabolismo , ARN Polimerasas Dirigidas por ADN/química , Factores de Transcripción/química
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