RESUMO
RNA polymerase (RNAP) frequently pauses during the transcription of DNA to RNA to regulate gene expression. Transcription factors NusA and NusG modulate pausing, have opposing roles, but can bind RNAP simultaneously. Here we report cryo-EM reconstructions of Escherichia coli RNAP bound to NusG, or NusA, or both. RNAP conformational changes, referred to as swivelling, correlate with transcriptional pausing. NusA facilitates RNAP swivelling to further increase pausing, while NusG counteracts this role. Their structural effects are consistent with biochemical results on two categories of transcriptional pauses. In addition, the structures suggest a cooperative mechanism of NusA and NusG during Rho-mediated transcription termination. Our results provide a structural rationale for the stochastic nature of pausing and termination and how NusA and NusG can modulate it.
Assuntos
Proteínas de Escherichia coli , Fatores de Transcrição , Proteínas de Bactérias/metabolismo , RNA Polimerases Dirigidas por DNA/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Conformação de Ácido Nucleico , Fatores de Alongamento de Peptídeos/metabolismo , Fatores de Transcrição/metabolismo , Transcrição Gênica , Fatores de Elongação da Transcrição/metabolismoRESUMO
Bromodomain-containing protein 4 (BRD4) is an epigenetic reader and oncology drug target that regulates gene transcription through binding to acetylated chromatin via bromodomains. Phosphorylation by casein kinase II (CK2) regulates BRD4 function, is necessary for active transcription and is involved in resistance to BRD4 drug inhibition in triple-negative breast cancer. Here, we provide the first biophysical analysis of BRD4 phospho-regulation. Using integrative structural biology, we show that phosphorylation by CK2 modulates the dimerization of human BRD4. We identify two conserved regions, a coiled-coil motif and the Basic-residue enriched Interaction Domain (BID), essential for the BRD4 structural rearrangement, which we term the phosphorylation-dependent dimerization domain (PDD). Finally, we demonstrate that bivalent inhibitors induce a conformational change within BRD4 dimers in vitro and in cancer cells. Our results enable the proposal of a model for BRD4 activation critical for the characterization of its protein-protein interaction network and for the development of more specific therapeutics.
Assuntos
Proteínas de Ciclo Celular/genética , Regulação da Expressão Gênica , Fatores de Transcrição/genética , Caseína Quinase II/genética , Caseína Quinase II/metabolismo , Proteínas de Ciclo Celular/metabolismo , Humanos , Fosforilação , Fatores de Transcrição/metabolismoRESUMO
Regulatory sequences or erroneous incorporations during DNA transcription cause RNA polymerase backtracking and inactivation in all kingdoms of life. Reactivation requires RNA transcript cleavage. Essential transcription factors (GreA and GreB, or TFIIS) accelerate this reaction. We report four cryo-EM reconstructions of Escherichia coli RNA polymerase representing the entire reaction pathway: (1) a backtracked complex; a backtracked complex with GreB (2) before and (3) after RNA cleavage; and (4) a reactivated, substrate-bound complex with GreB before RNA extension. Compared with eukaryotes, the backtracked RNA adopts a different conformation. RNA polymerase conformational changes cause distinct GreB states: a fully engaged GreB before cleavage; a disengaged GreB after cleavage; and a dislodged, loosely bound GreB removed from the active site to allow RNA extension. These reconstructions provide insight into the catalytic mechanism and dynamics of RNA cleavage and extension and suggest how GreB targets backtracked complexes without interfering with canonical transcription.
Assuntos
RNA Polimerases Dirigidas por DNA/química , Proteínas de Escherichia coli/química , Complexos Multiproteicos/química , RNA/química , Transcrição Gênica , Fatores de Elongação da Transcrição/química , Sequência de Aminoácidos/genética , Domínio Catalítico/genética , Microscopia Crioeletrônica , RNA Polimerases Dirigidas por DNA/genética , Escherichia coli/química , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Complexos Multiproteicos/genética , Ligação Proteica , Conformação Proteica , RNA/genética , Clivagem do RNA/genética , Motivos de Ligação ao RNA/genética , Fatores de Transcrição/química , Fatores de Transcrição/genética , Fatores de Elongação da Transcrição/genéticaRESUMO
Transcriptional pausing by RNA polymerases (RNAPs) is a key mechanism to regulate gene expression in all kingdoms of life and is a prerequisite for transcription termination. The essential bacterial transcription factor NusA stimulates both pausing and termination of transcription, thus playing a central role. Here, we report single-particle electron cryo-microscopy reconstructions of NusA bound to paused E. coli RNAP elongation complexes with and without a pause-enhancing hairpin in the RNA exit channel. The structures reveal four interactions between NusA and RNAP that suggest how NusA stimulates RNA folding, pausing, and termination. An asymmetric translocation intermediate of RNA and DNA converts the active site of the enzyme into an inactive state, providing a structural explanation for the inhibition of catalysis. Comparing RNAP at different stages of pausing provides insights on the dynamic nature of the process and the role of NusA as a regulatory factor.