RESUMO
In every domain of life, NusG-like proteins bind to the elongating RNA polymerase (RNAP) to support processive RNA synthesis and to couple transcription to ongoing cellular processes. Structures of factor-bound transcription elongation complexes (TECs) reveal similar contacts to RNAP, consistent with a shared mechanism of action. However, NusG homologs differ in their regulatory roles, modes of recruitment, and effects on RNA synthesis. Some of these differences could be due to conformational changes in RNAP and NusG-like proteins, which cannot be captured in static structures. Here, we employed hydrogen-deuterium exchange mass spectrometry to investigate changes in local and non-local structural dynamics of Escherichia coli NusG and its paralog RfaH, which have opposite effects on expression of xenogenes, upon binding to TEC. We found that NusG and RfaH regions that bind RNAP became solvent-protected in factor-bound TECs, whereas RNAP regions that interact with both factors showed opposite deuterium uptake changes when bound to NusG or RfaH. Additional changes far from the factor-binding site were observed only with RfaH. Our results provide insights into differences in structural dynamics exerted by NusG and RfaH during binding to TEC, which may explain their different functional outcomes and allosteric regulation of transcriptional pausing by RfaH.
Assuntos
Proteínas de Escherichia coli , Fatores de Alongamento de Peptídeos , Transativadores , Transcrição Gênica , Sítios de Ligação , RNA Polimerases Dirigidas por DNA/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Fatores de Alongamento de Peptídeos/metabolismo , RNA/metabolismo , Transativadores/metabolismo , Fatores de Elongação da Transcrição/metabolismoAssuntos
Subunidade alfa 2 de Fator de Ligação ao Core/genética , Proteínas de Ligação a DNA/genética , Regulação Leucêmica da Expressão Gênica , Histona-Lisina N-Metiltransferase/genética , Proteína de Leucina Linfoide-Mieloide/genética , Proteínas de Fusão Oncogênica/genética , Osteopontina/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Fatores de Elongação da Transcrição/genética , Processamento Alternativo , Linfócitos B/metabolismo , Linfócitos B/patologia , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Criança , Pré-Escolar , Subunidade alfa 2 de Fator de Ligação ao Core/metabolismo , Proteínas de Ligação a DNA/metabolismo , Feminino , Histona-Lisina N-Metiltransferase/metabolismo , Humanos , Lactente , Masculino , Proteína de Leucina Linfoide-Mieloide/metabolismo , Proteínas de Fusão Oncogênica/metabolismo , Osteopontina/antagonistas & inibidores , Osteopontina/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras/diagnóstico , Leucemia-Linfoma Linfoblástico de Células Precursoras/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras/patologia , Prognóstico , Isoformas de Proteínas/antagonistas & inibidores , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Fatores de Risco , Fatores de Elongação da Transcrição/metabolismoRESUMO
Light makes carbon fixation possible, allowing plant and animal life on Earth. We have previously shown that light regulates alternative splicing in plants. Light initiates a chloroplast retrograde signaling that regulates nuclear alternative splicing of a subset of Arabidopsis thaliana transcripts. Here, we show that light promotes RNA polymerase II (Pol II) elongation in the affected genes, whereas in darkness, elongation is lower. These changes in transcription are consistent with elongation causing the observed changes in alternative splicing, as revealed by different drug treatments and genetic evidence. The light control of splicing and elongation is abolished in an Arabidopsis mutant defective in the transcription factor IIS (TFIIS). We report that the chloroplast control of nuclear alternative splicing in plants responds to the kinetic coupling mechanism found in mammalian cells, providing unique evidence that coupling is important for a whole organism to respond to environmental cues.