RESUMEN
A complete, 52-protein, 2.5 million dalton, Mediator-RNA polymerase II pre-initiation complex (Med-PIC) was assembled and analyzed by cryo-electron microscopy and by chemical cross-linking and mass spectrometry. The resulting complete Med-PIC structure reveals two components of functional significance, absent from previous structures, a protein kinase complex and the Mediator-activator interaction region. It thereby shows how the kinase and its target, the C-terminal domain of the polymerase, control Med-PIC interaction and transcription.
Asunto(s)
Complejo Mediador/química , Complejo Mediador/metabolismo , Modelos Moleculares , ARN Polimerasa II/química , ARN Polimerasa II/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/enzimología , Saccharomyces cerevisiae/genética , Microscopía por Crioelectrón , Regulación de la Expresión Génica , Espectrometría de Masas , Fosforilación , Estructura Terciaria de Proteína , Subunidades de Proteína/química , Subunidades de Proteína/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMEN
Mediator is a universal adaptor for transcription control. It serves as an interface between gene-specific activator or repressor proteins and the general RNA polymerase II (pol II) transcription machinery. Previous structural studies revealed a relatively small part of Mediator and none of the gene activator-binding regions. We have determined the cryo-EM structure of the Mediator at near-atomic resolution. The structure reveals almost all amino acid residues in ordered regions, including the major targets of activator proteins, the Tail module, and the Med1 subunit of the Middle module. Comparison of Mediator structures with and without pol II reveals conformational changes that propagate across the entire Mediator, from Head to Tail, coupling activator- and pol II-interacting regions.
Asunto(s)
Subunidad 1 del Complejo Mediador/metabolismo , Aminoácidos/genética , Conformación Proteica , ARN Polimerasa II/metabolismo , Factores de Transcripción/metabolismo , Transcripción Genética/genéticaRESUMEN
Whereas RNA polymerase II (Pol II) transcription start sites (TSSs) occur about 30-35 bp downstream of the TATA box in metazoans, TSSs are located 40-120 bp downstream in S. cerevisiae. Promoter melting begins about 12 bp downstream in all eukaryotes, so Pol II is presumed to "scan" further downstream before starting transcription in yeast. Here we report that removal of the kinase complex TFIIK from TFIIH shifts the TSS in a yeast system upstream to the location observed in metazoans. Conversely, moving the normal TSS to an upstream location enables a high level of TFIIK-independent transcription in the yeast system. We distinguish two stages of the transcription initiation process: bubble formation by TFIIH, which fills the Pol II active center with single-stranded DNA, and subsequent scanning downstream, also driven by TFIIH, which requires displacement of the initial bubble. Omission of TFIIK uncouples the two stages of the process.
Asunto(s)
ARN Polimerasa II/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Factor de Transcripción TFIIH/genética , Sitio de Iniciación de la Transcripción/fisiología , Secuencia de Bases , Conformación de Ácido Nucleico , Regiones Promotoras Genéticas , Proteínas de Saccharomyces cerevisiae/metabolismo , Factor de Transcripción TFIIH/metabolismo , Transcripción Genética/genéticaRESUMEN
Chromatin isolated from the chromosomal locus of the PHO5 gene of yeast in a transcriptionally repressed state was transcribed with 12 pure proteins (80 polypeptides): RNA polymerase II, six general transcription factors, TFIIS, the Pho4 gene activator protein, and the SAGA, SWI/SNF, and Mediator complexes. Contrary to expectation, a nucleosome occluding the TATA box and transcription start sites did not impede transcription but rather, enhanced it: the level of chromatin transcription was at least sevenfold greater than that of naked DNA, and chromatin gave patterns of transcription start sites closely similar to those occurring in vivo, whereas naked DNA gave many aberrant transcripts. Both histone acetylation and trimethylation of H3K4 (H3K4me3) were important for chromatin transcription. The nucleosome, long known to serve as a general gene repressor, thus also performs an important positive role in transcription.
Asunto(s)
Regulación Fúngica de la Expresión Génica , Nucleosomas/genética , Transcripción Genética , Acetilación , Fosfatasa Ácida/genética , Secuencia de Bases , ADN Circular/genética , ADN de Hongos/genética , Histonas/metabolismo , Metilación , Complejos Multiproteicos , Regiones Promotoras Genéticas/genética , Procesamiento Proteico-Postraduccional , ARN de Hongos/genética , ARN Mensajero/biosíntesis , ARN Mensajero/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Análisis de Secuencia de ARN , Factores de Transcripción/metabolismoRESUMEN
General transcription factor TFIIH, previously described as a 10-subunit complex, is essential for transcription and DNA repair. An eleventh subunit now identified, termed Tfb6, exhibits 45% sequence similarity to human nuclear mRNA export factor 5. Tfb6 dissociates from TFIIH as a heterodimer with the Ssl2 subunit, a DNA helicase that drives promoter melting for the initiation of transcription. Tfb6 does not, however, dissociate Ssl2 from TFIIH in the context of a fully assembled transcription preinitiation complex. Our findings suggest a dynamic state of Ssl2, allowing its engagement in multiple cellular processes.
Asunto(s)
ADN Helicasas/metabolismo , Subunidades de Proteína/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimología , Saccharomyces cerevisiae/genética , Factor de Transcripción TFIIH/metabolismo , Factores de Transcripción/metabolismo , Transcripción Genética , Cromatografía Liquida , Eliminación de Gen , Regulación Fúngica de la Expresión Génica/efectos de la radiación , Humanos , Espectrometría de Masas , Fenotipo , Fosforilación/efectos de la radiación , Unión Proteica/efectos de la radiación , ARN Mensajero/genética , ARN Mensajero/metabolismo , Saccharomyces cerevisiae/efectos de la radiación , Proteínas de Saccharomyces cerevisiae/química , Temperatura , Factor de Transcripción TFIIH/química , Factores de Transcripción/química , Transcripción Genética/efectos de la radiación , Rayos UltravioletaRESUMEN
Whereas individual RNA polymerase II (pol II)-general transcription factor (GTF) complexes are unstable, an assembly of pol II with six GTFs and promoter DNA could be isolated in abundant homogeneous form. The resulting complete pol II transcription preinitiation complex (PIC) contained equimolar amounts of all 31 protein components. An intermediate in assembly, consisting of four GTFs and promoter DNA, could be isolated and supplemented with the remaining components for formation of the PIC. Nuclease digestion and psoralen cross-linking mapped the PIC between positions -70 and -9, centered on the TATA box. Addition of ATP to the PIC resulted in quantitative conversion to an open complex, which retained all 31 proteins, contrary to expectation from previous studies. Addition of the remaining NTPs resulted in run-off transcription, with an efficiency that was promoter-dependent and was as great as 17.5% with the promoters tested.
Asunto(s)
ADN de Hongos/química , Regiones Promotoras Genéticas/fisiología , ARN Polimerasa II/química , Saccharomyces cerevisiae/enzimología , Factores de Transcripción/química , Transcripción Genética/fisiología , ADN de Hongos/metabolismo , ARN Polimerasa II/metabolismo , Factores de Transcripción/metabolismoRESUMEN
RNA-dependent RNA polymerases play a vital role in the growth of RNA viruses where they are responsible for genome replication, but do so with rather low fidelity that allows for the rapid adaptation to different host cell environments. These polymerases are also a target for antiviral drug development. However, both drug discovery efforts and our understanding of fidelity determinants have been hampered by a lack of detailed structural information about functional polymerase-RNA complexes and the structural changes that take place during the elongation cycle. Many of the molecular details associated with nucleotide selection and catalysis were revealed in our recent structure of the poliovirus polymerase-RNA complex solved by first purifying and then crystallizing stalled elongation complexes. In the work presented here we extend that basic methodology to determine nine new structures of poliovirus, coxsackievirus, and rhinovirus elongation complexes at 2.2-2.9 Å resolution. The structures highlight conserved features of picornaviral polymerases and the interactions they make with the template and product RNA strands, including a tight grip on eight basepairs of the nascent duplex, a fully pre-positioned templating nucleotide, and a conserved binding pocket for the +2 position template strand base. At the active site we see a pre-bound magnesium ion and there is conservation of a non-standard backbone conformation of the template strand in an interaction that may aid in triggering RNA translocation via contact with the conserved polymerase motif B. Moreover, by engineering plasticity into RNA-RNA contacts, we obtain crystal forms that are capable of multiple rounds of in-crystal catalysis and RNA translocation. Together, the data demonstrate that engineering flexible RNA contacts to promote crystal lattice formation is a versatile platform that can be used to solve the structures of viral RdRP elongation complexes and their catalytic cycle intermediates.
Asunto(s)
Enterovirus Humano B/enzimología , Modelos Moleculares , Poliovirus/enzimología , Conformación Proteica , ARN Polimerasa Dependiente del ARN/química , ARN/química , Rhinovirus/enzimología , Sitios de Unión/genética , Cristalización , Ingeniería Genética/métodos , ARN/metabolismo , ARN Polimerasa Dependiente del ARN/metabolismoRESUMEN
The structure of the general transcription factor IIB (TFIIB) in a complex with RNA polymerase II reveals three features crucial for transcription initiation: an N-terminal zinc ribbon domain of TFIIB that contacts the "dock" domain of the polymerase, near the path of RNA exit from a transcribing enzyme; a "finger" domain of TFIIB that is inserted into the polymerase active center; and a C-terminal domain, whose interaction with both the polymerase and with a TATA box-binding protein (TBP)-promoter DNA complex orients the DNA for unwinding and transcription. TFIIB stabilizes an early initiation complex, containing an incomplete RNA-DNA hybrid region. It may interact with the template strand, which sets the location of the transcription start site, and may interfere with RNA exit, which leads to abortive initiation or promoter escape. The trajectory of promoter DNA determined by the C-terminal domain of TFIIB traverses sites of interaction with TFIIE, TFIIF, and TFIIH, serving to define their roles in the transcription initiation process.