RESUMO
Structured RNAs and RNA-protein complexes (RNPs) fold through complex pathways that are replete with misfolded traps, and many RNAs and RNPs undergo extensive conformational changes during their functional cycles. These folding steps and conformational transitions are frequently promoted by RNA chaperone proteins, notably by superfamily 2 (SF2) RNA helicase proteins. The two largest families of SF2 helicases, DEAD-box and DEAH-box proteins, share evolutionarily conserved helicase cores, but unwind RNA helices through distinct mechanisms. Recent studies have advanced our understanding of how their distinct mechanisms enable DEAD-box proteins to disrupt RNA base pairs on the surfaces of structured RNAs and RNPs, while some DEAH-box proteins are adept at disrupting base pairs in the interior of RNPs. Proteins from these families use these mechanisms to chaperone folding and promote rearrangements of structured RNAs and RNPs, including the spliceosome, and may use related mechanisms to maintain cellular messenger RNAs in unfolded or partially unfolded conformations.
Assuntos
RNA Helicases/química , RNA/química , Ribonucleoproteínas/química , Conformação de Ácido Nucleico , Conformação ProteicaRESUMO
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
DEAD-box proteins are the largest family of nucleic acid helicases, and are crucial to RNA metabolism throughout all domains of life. They contain a conserved 'helicase core' of two RecA-like domains (domains (D)1 and D2), which uses ATP to catalyse the unwinding of short RNA duplexes by non-processive, local strand separation. This mode of action differs from that of translocating helicases and allows DEAD-box proteins to remodel large RNAs and RNA-protein complexes without globally disrupting RNA structure. However, the structural basis for this distinctive mode of RNA unwinding remains unclear. Here, structural, biochemical and genetic analyses of the yeast DEAD-box protein Mss116p indicate that the helicase core domains have modular functions that enable a novel mechanism for RNA-duplex recognition and unwinding. By investigating D1 and D2 individually and together, we find that D1 acts as an ATP-binding domain and D2 functions as an RNA-duplex recognition domain. D2 contains a nucleic-acid-binding pocket that is formed by conserved DEAD-box protein sequence motifs and accommodates A-form but not B-form duplexes, providing a basis for RNA substrate specificity. Upon a conformational change in which the two core domains join to form a 'closed state' with an ATPase active site, conserved motifs in D1 promote the unwinding of duplex substrates bound to D2 by excluding one RNA strand and bending the other. Our results provide a comprehensive structural model for how DEAD-box proteins recognize and unwind RNA duplexes. This model explains key features of DEAD-box protein function and affords a new perspective on how the evolutionarily related cores of other RNA and DNA helicases diverged to use different mechanisms.
Assuntos
RNA Helicases DEAD-box/química , RNA Helicases DEAD-box/metabolismo , Conformação de Ácido Nucleico , RNA de Cadeia Dupla/química , RNA de Cadeia Dupla/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimologia , Adenosina Trifosfatases/química , Adenosina Trifosfatases/metabolismo , Trifosfato de Adenosina/metabolismo , Motivos de Aminoácidos , Sequência de Bases , Domínio Catalítico , Sequência Conservada , Cristalografia por Raios X , Evolução Molecular , Sequência Rica em GC/genética , Modelos Moleculares , Estrutura Terciária de Proteína , RNA de Cadeia Dupla/genética , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/metabolismo , Relação Estrutura-Atividade , Especificidade por SubstratoRESUMO
The Saccharomyces cerevisiae DEAD-box protein Mss116p is a general RNA chaperone that functions in splicing mitochondrial group I and group II introns. Recent X-ray crystal structures of Mss116p in complex with ATP analogs and single-stranded RNA show that the helicase core induces a bend in the bound RNA, as in other DEAD-box proteins, while a C-terminal extension (CTE) induces a second bend, resulting in RNA crimping. Here, we illuminate these structures by using high-throughput genetic selections, unigenic evolution, and analyses of in vivo splicing activity to comprehensively identify functionally important regions and permissible amino acid substitutions throughout Mss116p. The functionally important regions include those containing conserved sequence motifs involved in ATP and RNA binding or interdomain interactions, as well as previously unidentified regions, including surface loops that may function in protein-protein interactions. The genetic selections recapitulate major features of the conserved helicase motifs seen in other DEAD-box proteins but also show surprising variations, including multiple novel variants of motif III (SAT). Patterns of amino acid substitutions indicate that the RNA bend induced by the helicase core depends on ionic and hydrogen-bonding interactions with the bound RNA; identify a subset of critically interacting residues; and indicate that the bend induced by the CTE results primarily from a steric block. Finally, we identified two conserved regions-one the previously noted post II region in the helicase core and the other in the CTE-that may help displace or sequester the opposite RNA strand during RNA unwinding.
Assuntos
Motivos de Aminoácidos , RNA Helicases DEAD-box/genética , RNA Helicases DEAD-box/metabolismo , Splicing de RNA , RNA Fúngico/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Sequência de Aminoácidos , Sítios de Ligação , Northern Blotting , Sequência Conservada , Cristalografia por Raios X , RNA Helicases DEAD-box/química , Evolução Molecular , Immunoblotting , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Mutação/genética , Ligação Proteica , Conformação Proteica , Saccharomyces cerevisiae/crescimento & desenvolvimento , Proteínas de Saccharomyces cerevisiae/química , Homologia de Sequência de AminoácidosRESUMO
Triptolide (1) is a structurally unique diterpene triepoxide isolated from a traditional Chinese medicinal plant with anti-inflammatory, immunosuppressive, contraceptive and antitumor activities. Its molecular mechanism of action, however, has remained largely elusive to date. We report that triptolide covalently binds to human XPB (also known as ERCC3), a subunit of the transcription factor TFIIH, and inhibits its DNA-dependent ATPase activity, which leads to the inhibition of RNA polymerase II-mediated transcription and likely nucleotide excision repair. The identification of XPB as the target of triptolide accounts for the majority of the known biological activities of triptolide. These findings also suggest that triptolide can serve as a new molecular probe for studying transcription and, potentially, as a new type of anticancer agent through inhibition of the ATPase activity of XPB.
Assuntos
Antineoplásicos/farmacologia , Produtos Biológicos/farmacologia , DNA Helicases/metabolismo , Proteínas de Ligação a DNA/metabolismo , Diterpenos/farmacologia , Fenantrenos/farmacologia , Fator de Transcrição TFIIH/metabolismo , Adenosina Trifosfatases/química , Adenosina Trifosfatases/metabolismo , Antineoplásicos/química , Antineoplásicos/isolamento & purificação , Produtos Biológicos/química , Produtos Biológicos/isolamento & purificação , Sobrevivência Celular/efeitos dos fármacos , DNA Helicases/química , Proteínas de Ligação a DNA/química , Diterpenos/química , Diterpenos/isolamento & purificação , Relação Dose-Resposta a Droga , Medicamentos de Ervas Chinesas/química , Medicamentos de Ervas Chinesas/isolamento & purificação , Medicamentos de Ervas Chinesas/farmacologia , Compostos de Epóxi/química , Compostos de Epóxi/isolamento & purificação , Compostos de Epóxi/farmacologia , Células HeLa , Humanos , Fenantrenos/química , Fenantrenos/isolamento & purificação , Ligação Proteica , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo , RNA Polimerase II/genética , RNA Polimerase II/metabolismo , Fator de Transcrição TFIIH/químicaRESUMO
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.
Assuntos
Regiões Promotoras Genéticas/genética , RNA Polimerase II/metabolismo , Fatores Associados à Proteína de Ligação a TATA/química , Fatores Associados à Proteína de Ligação a TATA/metabolismo , Fator de Transcrição TFIID/metabolismo , DNA/química , DNA/metabolismo , Pegada de DNA , Desoxirribonuclease I/metabolismo , Humanos , Isomerismo , Cinética , Modelos Biológicos , Conformação de Ácido Nucleico , Ligação Proteica , Fator de Transcrição TFIIA/metabolismo , Fatores de Transcrição TFII/metabolismo , Sítio de Iniciação de Transcrição , Transcrição GênicaRESUMO
To better understand the mechanism of steps in early transcription by RNA polymerase II (pol II), we investigated the molecular determinants of transcript slipping within complexes assembled on promoters containing a pre-melted transcription bubble from -9 to +3. Transcript slippage occurs when an RNA transcript contains a repetitive sequence that allows the transcript to slip back and pair with the template strand of the DNA at a new register before transcription continues. We established the contributions of individual transcription factors, DNA elements, and RNA length to slipping on a heteroduplex template using a highly purified human pol II transcription system. We found that transcripts slip at a very defined point in the transcription reaction, after pol II completes phosphodiester bond synthesis at register +5. This point is set by the position of the polymerase active site on the DNA template, as opposed to the length of the transcript, as well as by a repetitive CUCU sequence that must occur from +2 to +5. Interestingly, slipping at this juncture is induced by TATA-binding protein and transcription factor IIB and requires a TATA box but not a transcription factor IIB recognition sequence. We propose a model in which transcribing complexes, upon completing phosphodiester bond synthesis at register +5, enter one of two branches in which they either complete productive synthesis of the transcript or undergo multiple rounds of transcript slipping.