RESUMO
RNA polymerase II (Pol II) is a well-characterized DNA-dependent RNA polymerase, which has also been reported to have RNA-dependent RNA polymerase (RdRP) activity. Natural cellular RNA substrates of mammalian Pol II, however, have not been identified and the cellular function of the Pol II RdRP activity is unknown. We found that Pol II can use a non-coding RNA, B2 RNA, as both a substrate and a template for its RdRP activity. Pol II extends B2 RNA by 18 nt on its 3'-end in an internally templated reaction. The RNA product resulting from extension of B2 RNA by the Pol II RdRP can be removed from Pol II by a factor present in nuclear extracts. Treatment of cells with α-amanitin or actinomycin D revealed that extension of B2 RNA by Pol II destabilizes the RNA. Our studies provide compelling evidence that mammalian Pol II acts as an RdRP to control the stability of a cellular RNA by extending its 3'-end.
Assuntos
RNA Polimerase II/fisiologia , Estabilidade de RNA , RNA não Traduzido/genética , RNA não Traduzido/metabolismo , Elongação da Transcrição Genética , Animais , Sequência de Bases , Células HeLa , Humanos , Camundongos , Modelos Biológicos , Dados de Sequência Molecular , Células NIH 3T3 , Conformação de Ácido Nucleico , RNA Polimerase II/genética , RNA Polimerase II/metabolismo , Estabilidade de RNA/genética , Estabilidade de RNA/fisiologia , RNA Polimerase Dependente de RNA/genética , RNA Polimerase Dependente de RNA/metabolismo , RNA Polimerase Dependente de RNA/fisiologia , Transcrição Gênica/genéticaRESUMO
Mutations in ribosomal RNA (rRNA) have traditionally been detected by the primer extension assay, which is a tedious and multistage procedure. Here, we describe a simple and straightforward fluorescence assay based on binary deoxyribozyme (BiDz) sensors. The assay uses two short DNA oligonucleotides that hybridize specifically to adjacent fragments of rRNA, one of which contains a mutation site. This hybridization results in the formation of a deoxyribozyme catalytic core that produces the fluorescent signal and amplifies it due to multiple rounds of catalytic action. This assay enables us to expedite semi-quantification of mutant rRNA content in cell cultures starting from whole cells, which provides information useful for optimization of culture preparation prior to ribosome isolation. The method requires less than a microliter of a standard Escherichia coli cell culture and decreases analysis time from several days (for primer extension assay) to 1.5 h with hands-on time of â¼10 min. It is sensitive to single-nucleotide mutations. The new assay simplifies the preliminary analysis of RNA samples and cells in molecular biology and cloning experiments and is promising in other applications where fast detection/quantification of specific RNA is required.
Assuntos
Técnicas Biossensoriais , DNA Catalítico/metabolismo , Mutação , RNA Bacteriano/genética , RNA Ribossômico/genética , Sequência de Bases , Escherichia coli/genética , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Hibridização de Ácido Nucleico , RNA Bacteriano/química , RNA Ribossômico/químicaRESUMO
UNLABELLED: Lytic infection by herpes simplex virus 1 (HSV-1) triggers a change in many host cell programs as the virus strives to express its own genes and replicate. Part of this process is repression of host cell transcription by RNA polymerase II (Pol II), which also transcribes the viral genome. Here, we describe a global characterization of Pol II occupancy on the viral and host genomes in response to HSV-1 infection using chromatin immunoprecipitation followed by deep sequencing (ChIP-seq). The data reveal near-complete loss of Pol II occupancy throughout host cell mRNA genes, in both their bodies and promoter-proximal regions. Increases in Pol II occupancy of host cell genes, which would be consistent with robust transcriptional activation, were not observed. HSV-1 infection induced a more potent and widespread repression of Pol II occupancy than did heat shock, another cellular stress that widely represses transcription. Concomitant with the loss of host genome Pol II occupancy, we observed Pol II covering the HSV-1 genome, reflecting a high level of viral gene transcription. Interestingly, the positions of the peaks of Pol II occupancy at HSV-1 and host cell promoters were different. IMPORTANCE: We investigated the effect of herpes simplex virus 1 (HSV-1) infection on transcription of host cell and viral genes by RNA polymerase II (Pol II). The approach we used was to determine how levels of genome-bound Pol II changed after HSV-1 infection. We found that HSV-1 caused a profound loss of Pol II occupancy across the host cell genome. Increases in Pol II occupancy were not observed, showing that no host genes were activated after infection. In contrast, Pol II occupied the entire HSV-1 genome. Moreover, the pattern of Pol II at HSV-1 genes differed from that on host cell genes, suggesting a unique mode of viral gene transcription. These studies provide new insight into how HSV-1 causes changes in the cellular program of gene expression and how the virus coopts host Pol II for its own use.
Assuntos
DNA/metabolismo , Herpesvirus Humano 1/fisiologia , Interações Hospedeiro-Patógeno , RNA Polimerase II/metabolismo , Replicação Viral , Animais , Linhagem Celular , Imunoprecipitação da Cromatina , Herpesvirus Humano 1/crescimento & desenvolvimento , Sequenciamento de Nucleotídeos em Larga Escala , Camundongos , Regiões Promotoras Genéticas , Ligação Proteica , Análise de Sequência de DNARESUMO
Proteins which bind to nucleic acids and regulate their structure and functions are numerous and exceptionally important. Such proteins employ a variety of strategies for recognition of the relevant structural elements in their nucleic acid substrates, some of which have been shown to involve rather subtle interactions which might have been difficult to design from first principles. In the present study, we have explored the preparation of proteins containing unnatural amino acids having nucleobase side chains. In principle, the introduction of multiple nucleobase amino acids into the nucleic acid binding domain of a protein should enable these modified proteins to interact with their nucleic acid substrates using Watson-Crick and other base pairing interactions. We describe the synthesis of five alanyl nucleobase amino acids protected in a fashion which enabled their attachment to a suppressor tRNA, and their incorporation into each of two proteins with acceptable efficiencies. The nucleobases studied included cytosine, uracil, thymine, adenine and guanine, i.e. the major nucleobase constituents of DNA and RNA. Dihydrofolate reductase was chosen as one model protein to enable direct comparison of the facility of incorporation of the nucleobase amino acids with numerous other unnatural amino acids studied previously. The Klenow fragment of DNA polymerase I was chosen as a representative DNA binding protein whose mode of action has been studied in detail.
Assuntos
Alanina/análogos & derivados , Alanina/síntese química , Purinas/síntese química , Pirimidinonas/síntese química , Tetra-Hidrofolato Desidrogenase/química , Alanina/genética , Códon , DNA Polimerase I/genética , Escherichia coli , RNA Mensageiro/química , RNA Mensageiro/genética , RNA de Transferência/síntese química , RNA de Transferência/genética , Tetra-Hidrofolato Desidrogenase/genética , Aminoacilação de RNA de TransferênciaRESUMO
Described herein are the syntheses and photophysical characterization of three novel cyanotryptophans, and their efficient incorporation into proteins as fluorescent probes. Photophysical characteristics indicated that each was significantly brighter and red-shifted in fluorescence emission relative to tryptophan. Each analogue was used to activate a suppressor tRNA transcript and was incorporated with good efficiency into two different positions (Trp22 and Trp74) of Escherichia coli dihydrofolate reductase (ecDHFR). The Trp analogues could be monitored selectively in the presence of multiple native Trp residues in DHFR. 6-CNTrp (A) formed an efficient Förster resonance energy transfer (FRET) pair with l-(7-hydroxycoumarin-4-yl)ethylglycine (HCO, D) at position 17. Further, 6-CNTrp (A) was incorporated into two DNA binding proteins, including the Klenow fragment of DNA polymerase I and an RNA recognition motif (RRM2) of heterogeneous nuclear ribonucleoprotein L-like (hnRNP LL). Using these proteins, we demonstrated the use of FRET involving A as a fluorescence donor and benzo[g]quinazoline-2,4-(1H,3H)-dione 2'-deoxyriboside (Tf) or 4-aminobenzo[g]quinazoline-2-one 2'-deoxyriboside (Cf) as fluorescent acceptors to study the binding interaction of the Klenow fragment with duplex DNA oligomers (labeled with Tf), or the domain-specific association between hnRNP LL and the BCL2 i-motif DNA (labeled with Cf). Thus, the non-natural amino acid could be used as a FRET partner for studying protein-nucleic acid interactions. Together, these findings demonstrate the potential utility of 6-CNTrp (A) as a fluorescence donor for the study of protein conformational events.
Assuntos
DNA/química , Corantes Fluorescentes/química , Proteínas/química , Triptofano/análogos & derivados , Transferência Ressonante de Energia de Fluorescência , Ligação Proteica , Conformação Proteica , Triptofano/químicaRESUMO
Noncoding RNAs (ncRNAs) are now recognized as transregulators of eukaryotic transcription, a role once attributed exclusively to protein factors. Two ncRNAs in mammalian cells have been shown to repress general mRNA transcription by RNA polymerase II (Pol II) in response to heat shock: mouse B2 RNA and human Alu RNA. B2 and Alu RNAs bind directly and tightly to Pol II and co-occupy the promoters of repressed genes along with the polymerase. Here, we identified the molecular mechanism by which mouse B2 RNA and human Alu RNA repress Pol II transcription. Biochemical assays to probe the network of protein-DNA interactions at the promoter revealed that B2 and Alu RNAs prevent Pol II from establishing contacts with the promoter both upstream and downstream of the TATA box during closed complex formation. Disruption of these contacts correlates with transcriptional repression. We conclude that B2 and Alu RNA prevent Pol II from properly engaging the DNA during closed complex formation, resulting in complexes with an altered conformation that are transcriptionally inert. In the absence of its normal contacts with the promoter, Pol II is likely held in these inactive complexes on DNA through interactions with promoter-bound TATA box-binding protein and transcription factor IIB.
Assuntos
Regiões Promotoras Genéticas/genética , RNA Polimerase II/genética , RNA não Traduzido/fisiologia , Transcrição Gênica , Animais , Regulação para Baixo/genética , Resposta ao Choque Térmico , Humanos , Camundongos , Ligação Proteica , RNA Polimerase II/metabolismo , Proteína de Ligação a TATA-Box , Fator de Transcrição TFIIBRESUMO
Gene transcription is regulated in response to environmental changes and developmental cues. In mammalian cells subjected to stress conditions such as heat shock, transcription of most protein-coding genes decreases, while the transcription of heat shock protein genes increases. Repression involves direct binding to RNA polymerase II (Pol II) of certain noncoding RNAs (ncRNAs) that are upregulated upon heat shock. Another class of ncRNAs is also upregulated and binds to Pol II but does not inhibit transcription. Incorporation of repressive ncRNAs into pre-initiation complexes prevents transcription initiation, while non-repressive ncRNAs are displaced from Pol II by TFIIF. Here, we present cryo-electron microscopy reconstructions of human Pol II in complex with six different ncRNAs from mouse and human. Our structures show that both repressive and non-repressive ncRNAs bind to a conserved binding site within the cleft of Pol II. The site, which is also shared with a previously characterized yeast aptamer, is close to the active center and, thus, in an ideal position to regulate transcription. Importantly, additional RNA elements extend flexibly beyond the docking site. We propose that the differences concerning the repressive activity of the ncRNAs analyzed must be due to the distinct character of these more unstructured, flexible segments of the RNA that emanate from the cleft.
Assuntos
Repressão Epigenética , RNA Polimerase II/genética , RNA não Traduzido/genética , Fatores de Transcrição TFII/genética , Animais , Sítios de Ligação/genética , Microscopia Crioeletrônica , Células HeLa , Resposta ao Choque Térmico/genética , Humanos , Processamento de Imagem Assistida por Computador , Camundongos , Conformação Proteica , RNA Polimerase II/química , RNA não Traduzido/química , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Fatores de Transcrição TFII/química , Regulação para CimaRESUMO
Mouse B2 RNA represses RNA polymerase II (Pol II) transcription during the cellular heat shock response. B2 RNA binds Pol II, enters complexes at promoters, and keeps the polymerase from engaging the DNA. Here we show that phosphorylation of Ser5 residues in the Pol II carboxy terminal domain (CTD) decreases after heat shock at the promoter of the repressed actin gene in mouse cells, despite the continued presence of Cdk7 and cyclin H. Biochemical assays revealed that B2 RNA, when present with Pol II in promoter-bound complexes, specifically represses CTD phosphorylation by TFIIH.
Assuntos
RNA Polimerase II/metabolismo , RNA não Traduzido/metabolismo , Fator de Transcrição TFIIH/metabolismo , Actinas/genética , Animais , Sítios de Ligação , Western Blotting , Imunoprecipitação da Cromatina , Ciclina H/metabolismo , Quinases Ciclina-Dependentes/metabolismo , Temperatura Alta , Camundongos , Células NIH 3T3 , Fosforilação , Regiões Promotoras Genéticas/genética , Ligação Proteica , Proteínas Quinases/metabolismo , RNA não Traduzido/genética , Serina/metabolismo , Transcrição GênicaRESUMO
Transcription of mRNA genes requires that RNA polymerase II (Pol II) and the general transcription factors assemble on promoter DNA to form an organized complex capable of initiating transcription. Biochemical studies have shown that Pol II and TFIID (transcription factor IID) contact overlapping regions of the promoter, leading to the question of how these large factors reconcile their promoter interactions during complex assembly. To investigate how the TAF (TATA-binding protein-associated factor) subunits of TFIID alter the kinetic mechanism by which complexes assemble on promoters, we used a highly purified human transcription system. We found that TAFs sharply decrease the rate at which Pol II, TFIIB, and TFIIF assemble on promoter-bound TFIID-TFIIA. Interestingly, the slow step in this process is not recruitment of these factors to the DNA, but rather a postrecruitment isomerization of protein-DNA contacts that occurs throughout the core promoter. Our findings support a model in which Pol II and the general transcription factors rapidly bind promoter-bound TFIID-TFIIA, after which complexes undergo a slow isomerization in which the TAFs reorganize their contacts with the promoter to allow Pol II to properly engage the DNA. In this manner, TAFs kinetically repress basal transcription.