RESUMO
NusG, the only universally conserved transcription factor, comprises an N- and a C-terminal domain (NTD, CTD) that are flexibly connected and move independently in Escherichia coli and other organisms. In NusG from the hyperthermophilic bacterium Thermotoga maritima (tmNusG), however, NTD and CTD interact tightly. This closed state stabilizes the CTD, but masks the binding sites for the interaction partners Rho, NusE and RNA polymerase (RNAP), suggesting that tmNusG is autoinhibited. Furthermore, tmNusG and some other bacterial NusGs have an additional domain, DII, of unknown function. Here we demonstrate that tmNusG is indeed autoinhibited and that binding to RNAP may stabilize the open conformation. We identified two interdomain salt bridges as well as Phe336 as major determinants of the domain interaction. By successive weakening of this interaction we show that after domain dissociation tmNusG-CTD can bind to Rho and NusE, similar to the Escherichia coli NusG-CTD, indicating that these interactions are conserved in bacteria. Furthermore, we show that tmNusG-DII interacts with RNAP as well as nucleic acids with a clear preference for double stranded DNA. We suggest that tmNusG-DII supports tmNusG recruitment to the transcription elongation complex and stabilizes the tmNusG:RNAP complex, a necessary adaptation to high temperatures.
Assuntos
DNA Bacteriano/química , RNA Polimerases Dirigidas por DNA/química , Proteínas de Escherichia coli/química , Regulação Bacteriana da Expressão Gênica , Fatores de Alongamento de Peptídeos/química , Fator Rho/química , Thermotoga maritima/genética , Fatores de Transcrição/química , Sítios de Ligação , Sequência Conservada , DNA/química , DNA/genética , DNA/metabolismo , DNA Bacteriano/genética , DNA Bacteriano/metabolismo , RNA Polimerases Dirigidas por DNA/genética , RNA Polimerases Dirigidas por DNA/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Temperatura Alta , Fatores de Alongamento de Peptídeos/genética , Fatores de Alongamento de Peptídeos/metabolismo , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Estabilidade Proteica , Estrutura Secundária de Proteína , Fator Rho/genética , Fator Rho/metabolismo , Proteínas Ribossômicas/química , Proteínas Ribossômicas/genética , Proteínas Ribossômicas/metabolismo , Relação Estrutura-Atividade , Thermotoga maritima/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
In bacteria, RNA polymerase (RNAP), the central enzyme of transcription, is regulated by N-utilization substance (Nus) transcription factors. Several of these factors interact directly, and only transiently, with RNAP to modulate its function. As details of these interactions are largely unknown, we probed the RNAP binding surfaces of Escherichia coli (E. coli) Nus factors by nuclear magnetic resonance (NMR) spectroscopy. Perdeuterated factors with [(1)H,(13)C]-labeled methyl groups of Val, Leu, and Ile residues were titrated with protonated RNAP. After verification of this approach with the N-terminal domain (NTD) of NusG and RNAP we determined the RNAP binding site of NusE. It overlaps with the NusE interaction surface for the NusG C-terminal domain, indicating that RNAP and NusG compete for NusE and suggesting possible roles for the NusE:RNAP interaction, e.g. in antitermination and direct transcription:translation coupling. We solved the solution structure of NusA-NTD by NMR spectroscopy, identified its RNAP binding site with the same approach we used for NusG-NTD, and here present a detailed model of the NusA-NTD:RNAP:RNA complex.
Assuntos
Sítios de Ligação , RNA Polimerases Dirigidas por DNA/química , Ressonância Magnética Nuclear Biomolecular , Domínios e Motivos de Interação entre Proteínas , Fatores de Transcrição/química , RNA Polimerases Dirigidas por DNA/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Modelos Moleculares , Ligação Proteica , Biossíntese de Proteínas , Conformação Proteica , Soluções , Fatores de Transcrição/metabolismo , Transcrição GênicaRESUMO
RNA synthesis is a central process in all organisms, with RNA polymerase (RNAP) as the key enzyme. Multisubunit RNAPs are evolutionary related and are tightly regulated by a multitude of transcription factors. Although Escherichia coli RNAP has been studied extensively, only little information is available about its dynamics and transient interactions. This information, however, are crucial for the complete understanding of transcription regulation in atomic detail. To study RNAP by NMR spectroscopy we developed a highly efficient procedure for the assembly of active RNAP from separately expressed subunits that allows specific labeling of the individual constituents. We recorded [(1)H,(13)C] correlation spectra of isoleucine, leucine, and valine methyl groups of complete RNAP and the separately labeled ß' subunit within reconstituted RNAP. We further produced all RNAP subunits individually, established experiments to determine which RNAP subunit a certain regulator binds to, and identified the ß subunit to bind NusE.
Assuntos
RNA Polimerases Dirigidas por DNA/química , Espectroscopia de Ressonância Magnética , Sítios de Ligação , Espectroscopia de Ressonância Magnética Nuclear de Carbono-13 , RNA Polimerases Dirigidas por DNA/isolamento & purificação , RNA Polimerases Dirigidas por DNA/metabolismo , Escherichia coli/enzimologia , Modelos Moleculares , Ligação Proteica , Estrutura Secundária de Proteína , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo , Espectroscopia de Prótons por Ressonância Magnética , Proteínas Ribossômicas/química , Proteínas Ribossômicas/metabolismo , Fatores de Transcrição/química , Fatores de Transcrição/metabolismoRESUMO
NusG is a conserved regulatory protein interacting with RNA polymerase (RNAP) and other proteins to form multicomponent complexes that modulate transcription. The crystal structure of Thermotoga maritima NusG (TmNusG) shows a three-domain architecture, comprising well-conserved amino-terminal (NTD) and carboxy-terminal (CTD) domains with an additional, species-specific domain inserted into the NTD. NTD and CTD directly contact each other, occluding a surface of the NTD for binding to RNAP and a surface on the CTD interacting either with transcription termination factor Rho or transcription antitermination factor NusE. NMR spectroscopy confirmed the intramolecular NTD-CTD interaction up to the optimal growth temperature of Thermotoga maritima. The domain interaction involves a dynamic equilibrium between open and closed states and contributes significantly to the overall fold stability of the protein. Wild-type TmNusG and deletion variants could not replace endogenous Escherichia coli NusG, suggesting that the NTD-CTD interaction of TmNusG represents an autoinhibited state.