RESUMEN
The transition from transcription initiation to elongation is highly regulated in human cells but remains incompletely understood at the structural level. In particular, it is unclear how interactions between RNA polymerase II (RNA Pol II) and initiation factors are broken to enable promoter escape. Here, we reconstitute RNA Pol II promoter escape in vitro and determine high-resolution structures of initially transcribing complexes containing 8-, 10-, and 12-nt ordered RNAs and two elongation complexes containing 14-nt RNAs. We suggest that promoter escape occurs in three major steps. First, the growing RNA displaces the B-reader element of the initiation factor TFIIB without evicting TFIIB. Second, the rewinding of the transcription bubble coincides with the eviction of TFIIA, TFIIB, and TBP. Third, the binding of DSIF and NELF facilitates TFIIE and TFIIH dissociation, establishing the paused elongation complex. This three-step model for promoter escape fills a gap in our understanding of the initiation-elongation transition of RNA Pol II transcription.
Asunto(s)
Fosfoproteínas , Regiones Promotoras Genéticas , ARN Polimerasa II , Proteína de Unión a TATA-Box , Factor de Transcripción TFIIB , Factores de Transcripción , ARN Polimerasa II/metabolismo , ARN Polimerasa II/genética , Humanos , Factor de Transcripción TFIIB/metabolismo , Factor de Transcripción TFIIB/genética , Proteína de Unión a TATA-Box/metabolismo , Proteína de Unión a TATA-Box/genética , Factores de Transcripción/metabolismo , Factores de Transcripción/genética , Iniciación de la Transcripción Genética , Factor de Transcripción TFIIH/metabolismo , Factor de Transcripción TFIIH/genética , Factor de Transcripción TFIIH/química , Proteínas Nucleares/metabolismo , Proteínas Nucleares/genética , Unión Proteica , Factor de Transcripción TFIIA/metabolismo , Factor de Transcripción TFIIA/genética , Transcripción Genética , Elongación de la Transcripción Genética , ARN/metabolismo , ARN/genética , Factores de Transcripción TFII/metabolismo , Factores de Transcripción TFII/genéticaRESUMEN
Ca2+/calmodulin-dependent kinase IIα (CaMKIIα) is essential for synaptic plasticity and learning by decoding synaptic Ca2+ oscillations. Despite decades of extensive research, new mechanisms underlying CaMKIIα's function in synapses are still being discovered. Here, we discover that Shank3 is a specific binding partner for autoinhibited CaMKIIα. We demonstrate that Shank3 and GluN2B, via combined actions of Ca2+ and phosphatases, reciprocally bind to CaMKIIα. Under basal condition, CaMKIIα is recruited to the Shank3 subcompartment of postsynaptic density (PSD) via phase separation. Rise of Ca2+ concentration induces GluN2B-mediated recruitment of active CaMKIIα and formation of the CaMKIIα/GluN2B/PSD-95 condensates, which are autonomously dispersed upon Ca2+ removal. Protein phosphatases control the Ca2+-dependent shuttling of CaMKIIα between the two PSD subcompartments and PSD condensate formation. Activation of CaMKIIα further enlarges the PSD assembly and induces structural LTP. Thus, Ca2+-induced and phosphatase-checked shuttling of CaMKIIα between distinct PSD nano-domains can regulate phase separation-mediated PSD assembly and synaptic plasticity.
Asunto(s)
Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/metabolismo , Plasticidad Neuronal/fisiología , Fosfoproteínas Fosfatasas/metabolismo , Animales , Sitios de Unión , Calcio/metabolismo , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/química , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/genética , Homólogo 4 de la Proteína Discs Large/genética , Homólogo 4 de la Proteína Discs Large/metabolismo , Células HEK293 , Humanos , Ratones , Simulación del Acoplamiento Molecular , Proteínas del Tejido Nervioso/química , Proteínas del Tejido Nervioso/metabolismo , Fosforilación , Unión Proteica , Ratas , Receptores de N-Metil-D-Aspartato/genética , Receptores de N-Metil-D-Aspartato/metabolismo , Proteínas Asociadas a SAP90-PSD95/metabolismoRESUMEN
Dengue virus NS3 is a prototypical DEx(H/D) helicase that binds and hydrolyzes NTP to translocate along and unwind double-stranded nucleic acids. NS3 and NS4B are essential components of the flavivirus replication complex. Evidences showed that NS4B interacted with NS3 and modulated the helicase activity of NS3. Despite important insights into structural, mechanistic, and cellular aspects of the NS3 function, there is still a gap in understanding how it coordinates the helicase activities within the replicase complex for efficient replication. Here, using the DENV2 as a model, we redefined the critical region of NS4B required for NS3 function by pull-down and MST assays. The FRET-based unwinding assay showed that NS3 would accelerate unwinding duplex nucleic acids in the presence of NS4B (51-83). The simulated NS3-NS4B complex models based on the rigid-body docking delineated the potential interaction sites located in the conserved motif within the core domain of NS3. Mutations in motif I (I190A) and motif III (P319L) of NS3 interfered with the unwinding activity stimulated by NS4B. Upon binding to the NS3 helicase, NS4B assisted NS3 to dissociate from single-stranded nucleic acid and enabled NS3 helicase to keep high activity at high ATP concentrations. These results suggest that NS4B probably serves as an essential cofactor for NS3 to coordinate the ATP cycles and nucleic acid binding during viral genome replication.