ABSTRACT
Sequence-specific and programmable binding of proteins to RNA bears the potential to detect and manipulate target RNAs. Applications include analysis of subcellular RNA localization or post-transcriptional regulation but require sequence-specificity to be readily adjustable to any target RNA. The Pumilio homology domain binds an eight nucleotide target sequence in a predictable manner allowing for rational design of variants with new specificities. We describe a high-throughput system for screening Pumilio variants based on fluorescence-activated cell sorting of E. coli. Our approach should help optimizing variants obtained from rational design regarding folding and stability or identifying new variants with alternative binding modes.
Subject(s)
Escherichia coli/cytology , Flow Cytometry , Mutation , RNA-Binding Proteins/genetics , RNA-Binding Proteins/metabolism , RNA/genetics , RNA/metabolism , Base Sequence , Escherichia coli/metabolism , Protein Binding , Substrate SpecificityABSTRACT
Multicolor readout is an important feature of RNA detection techniques aiming at the investigation of RNA localization. Several detection methods have been developed, however they require either transfection of cells with the probe or prior tagging of the target RNA. We report a fully genetically encodable system for simultaneous detection of two RNAs by using green and yellow fluorescence based on tetramolecular fluorescence complementation (TetFC). To obtain yellow fluorescent protein (YFP), substitution T203Y was introduced into one of the three non-fluorescent GFP fragments; this was fused to different variants of the Homo sapiens Pumilio homology domain. Using different sets of fusion proteins we were able to discriminate between two closely related target RNAs based on the fluorescence signals at the respective wavelengths.
Subject(s)
RNA-Binding Proteins/metabolism , RNA/analysis , Sequence Analysis, RNA/methods , Spectrometry, Fluorescence , Transcription Factors/metabolism , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Base Sequence , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/metabolism , HeLa Cells , Humans , Luminescent Proteins/genetics , Luminescent Proteins/metabolism , Mutagenesis, Site-Directed , Protein Binding , Protein Engineering , RNA/metabolism , RNA-Binding Proteins/genetics , Recombinant Fusion Proteins/biosynthesis , Recombinant Fusion Proteins/chemistry , Transcription Factors/geneticsABSTRACT
BACKGROUND: Quorum sensing (QS) is a cell-to-cell communication system used by bacteria to regulate activities such as virulence, bioluminescence and biofilm formation. The most common QS signals in Gram-negative bacteria are N-acyl-homoserine lactones (AHLs). Aliivibrio salmonicida is the etiological agent of cold water vibriosis in Atlantic salmon, a disease which occurs mainly during seasons when the seawater is below 12°C. In this work we have constructed several mutants of A. salmonicida LFI1238 in order to study the LuxI/LuxR and AinS/AinR QS systems with respect to AHL production and biofilm formation. RESULTS: Using high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) we found that LuxI in A. salmonicida LFI1238 is responsible for producing seven of the different AHLs, whereas AinS is responsible for producing only one. The production of these various AHLs is dependent on both cell density and growth temperature. The AHLs were efficiently produced when wild type LFI1238 was grown at 6 or 12°C, however at 16°C AHL production decreased dramatically, and LFI1238 produced less than 5% of the maximum concentrations observed at 6°C. LitR, the master regulator of QS, was found to be a positive regulator of AinS-dependent AHL production, and to a lesser extent LuxI-dependent AHL production. This implies a connection between the two systems, and both systems were found to be involved in regulation of biofilm formation. Finally, inactivation of either luxR1 or luxR2 in the lux operon significantly reduced production of LuxI-produced AHLs. CONCLUSION: LuxI and AinS are the autoinducer synthases responsible for the eight AHLs in A. salmonicida. AHL production is highly dependent on growth temperature, and a significant decrease was observed when the bacterium was grown at a temperature above its limit for disease outbreak. Numerous AHLs could offer the opportunity for fine-tuning responses to changes in the environment.
Subject(s)
Acyl-Butyrolactones/metabolism , Aliivibrio salmonicida/enzymology , Aliivibrio salmonicida/radiation effects , Bacterial Proteins/metabolism , Aliivibrio salmonicida/genetics , Aliivibrio salmonicida/metabolism , Bacterial Proteins/genetics , Chromatography, High Pressure Liquid , Mutation , Tandem Mass Spectrometry , TemperatureABSTRACT
Ribonucleoprotein (RNP) complexes are widespread in nature and play crucial roles in gene regulation, RNA processing, and translation. Novel technologies, such as CRISPR-mediated genome engineering, stress the potential of RNP complexes to carry out complex tasks in molecular biology. Here we report a bottom-up approach for the programmable self-assembly of RNP complexes. The building blocks for RNP complex formation are RNAs and Pumilio proteins that can bind to RNA sequence-specifically. Correct RNP assembly triggers protein complementation of a tripartite GFP, thereby resulting in up to 25-fold increased fluorescence, and is strictly dependent on the correct RNA sequences. Our results indicate that Pumilio and guide RNAs are suitable building blocks for the correct self-assembly of RNP complexes consisting of up to six different components. Self-assembling RNP complexes might prove useful for complex biotechnological applications in RNA sensing, imaging, or processing.
