RESUMEN
Cell-free protein synthesis assays have become a valuable tool to understand transcriptional and translational processes. Here, we established a fluorescence-based coupled in vitro transcription-translation assay as a read-out system to simultaneously quantify mRNA and protein levels. We utilized the well-established quantification of the expression of shifted green fluorescent protein (sGFP) as a read-out of protein levels. In addition, we determined mRNA quantities using a fluorogenic Mango-(IV) RNA aptamer that becomes fluorescent upon binding to the fluorophore thiazole orange (TO). We utilized a Mango-(IV) RNA aptamer system comprising four subsequent Mango-(IV) RNA aptamer elements with improved sensitivity by building Mango arrays. The design of this reporter assay resulted in a sensitive read-out with a high signal-to-noise ratio, allowing us to monitor transcription and translation time courses in cell-free assays with continuous monitoring of fluorescence changes as well as snapshots of the reaction. Furthermore, we applied this dual read-out assay to investigate the function of thiamine-sensing riboswitches thiM and thiC from Escherichia coli and the adenine-sensing riboswitch ASW from Vibrio vulnificus and pbuE from Bacillus subtilis, which represent transcriptional and translational on- and off-riboswitches, respectively. This approach enabled a microplate-based application, a valuable addition to the toolbox for high-throughput screening of riboswitch function.
Asunto(s)
Aptámeros de Nucleótidos , Riboswitch , Adenina/química , Aptámeros de Nucleótidos/genética , Aptámeros de Nucleótidos/metabolismo , Fluorescencia , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Conformación de Ácido Nucleico , Sistema Libre de CélulasRESUMEN
The ribosomal S1 protein (rS1) is indispensable for translation initiation in Gram-negative bacteria. rS1 is a multidomain protein that acts as an RNA chaperone and ensures that mRNAs can bind the ribosome in a single-stranded conformation, which could be related to fast recognition. Although many ribosome structures were solved in recent years, a high-resolution structure of a two-domain mRNA-binding competent rS1 construct is not yet available. Here, we present the NMR solution structure of the minimal mRNA-binding fragment of Vibrio Vulnificus rS1 containing the domains D3 and D4. Both domains are homologues and adapt an oligonucleotide-binding fold (OB fold) motif. NMR titration experiments reveal that recognition of miscellaneous mRNAs occurs via a continuous interaction surface to one side of these structurally linked domains. Using a novel paramagnetic relaxation enhancement (PRE) approach and exploring different spin-labeling positions within RNA, we were able to track the location and determine the orientation of the RNA in the rS1-D34 bound form. Our investigations show that paramagnetically labeled RNAs, spiked into unmodified RNA, can be used as a molecular ruler to provide structural information on protein-RNA complexes. The dynamic interaction occurs on a defined binding groove spanning both domains with identical ß2-ß3-ß5 interfaces. Evidently, the 3'-ends of the cis-acting RNAs are positioned in the direction of the N-terminus of the rS1 protein, thus towards the 30S binding site and adopt a conformation required for translation initiation.
Asunto(s)
Proteínas Bacterianas/química , ARN Mensajero/química , Proteínas Ribosómicas/química , Vibrio vulnificus/química , Modelos Moleculares , Resonancia Magnética Nuclear Biomolecular , Unión Proteica , Biosíntesis de Proteínas , Dominios Proteicos , RiboswitchRESUMEN
Initiation of bacterial translation requires that the ribosome-binding site in mRNAs adopts single-stranded conformations. In Gram-negative bacteria the ribosomal protein S1 (rS1) is a key player in resolving of structured elements in mRNAs. However, the exact mechanism of how rS1 unfolds persistent secondary structures in the translation initiation region (TIR) is still unknown. Here, we show by NMR spectroscopy that Vibrio vulnificus rS1 displays a unique architecture of its mRNA-binding domains, where domains D3 and D4 provide the mRNA-binding platform and cover the nucleotide binding length of the full-length rS1. D5 significantly increases rS1's chaperone activity, although it displays structural heterogeneity both in isolation and in presence of the other domains, albeit to varying degrees. The heterogeneity is induced by the switch between the two equilibrium conformations and is triggered by an order-to-order transition of two mutually exclusive secondary structures (ß-strand-to-α-helix) of the 'AERERI' sequence. The conformational switching is exploited for melting of structured 5'-UTR's, as the conformational heterogeneity of D5 can compensate the entropic penalty of complex formation. Our data thus provides a detailed understanding of the intricate coupling of protein and RNA folding dynamics enabling translation initiation of structured mRNAs.
Asunto(s)
Proteínas Bacterianas/metabolismo , Conformación de Ácido Nucleico , Iniciación de la Cadena Peptídica Traduccional , Pliegue del ARN , ARN Mensajero/química , Proteínas Ribosómicas/metabolismo , Secuencia de Aminoácidos , Proteínas Bacterianas/genética , Secuencia de Bases , Sitios de Unión/genética , Escherichia coli/genética , Escherichia coli/metabolismo , ARN Bacteriano/genética , ARN Bacteriano/metabolismo , ARN Mensajero/genética , Proteínas Ribosómicas/genética , Ribosomas/genética , Ribosomas/metabolismo , Homología de Secuencia de Aminoácido , Vibrio vulnificus/genética , Vibrio vulnificus/metabolismoRESUMEN
The highly infectious disease COVID-19 caused by the Betacoronavirus SARS-CoV-2 poses a severe threat to humanity and demands the redirection of scientific efforts and criteria to organized research projects. The international COVID19-NMR consortium seeks to provide such new approaches by gathering scientific expertise worldwide. In particular, making available viral proteins and RNAs will pave the way to understanding the SARS-CoV-2 molecular components in detail. The research in COVID19-NMR and the resources provided through the consortium are fully disclosed to accelerate access and exploitation. NMR investigations of the viral molecular components are designated to provide the essential basis for further work, including macromolecular interaction studies and high-throughput drug screening. Here, we present the extensive catalog of a holistic SARS-CoV-2 protein preparation approach based on the consortium's collective efforts. We provide protocols for the large-scale production of more than 80% of all SARS-CoV-2 proteins or essential parts of them. Several of the proteins were produced in more than one laboratory, demonstrating the high interoperability between NMR groups worldwide. For the majority of proteins, we can produce isotope-labeled samples of HSQC-grade. Together with several NMR chemical shift assignments made publicly available on covid19-nmr.com, we here provide highly valuable resources for the production of SARS-CoV-2 proteins in isotope-labeled form.