Recombinant Escherichia coli O127:H6 tRNA pseudouridine synthase A (truA)

What is truA and what is its significance in E. coli O127:H6?

TruA (tRNA pseudouridine synthase A) is a highly conserved enzyme responsible for the modification of uridines to pseudouridines in tRNA molecules. In E. coli, including the O127:H6 strain, truA specifically modifies uridines at positions 38, 39, and/or 40 in the anticodon stem loop (ASL) of tRNAs . This modification is crucial for translational accuracy and efficiency, as it affects the stability and functionality of the tRNA molecules. E. coli O127:H6 is classified as an enteropathogenic E. coli (EPEC) strain that typically contains the H6 flagellar antigen . The recombinant truA protein from this strain serves as an important tool for studying RNA modification mechanisms and their biological implications.

How does truA from E. coli O127:H6 differ from other pseudouridine synthases?

TruA exhibits distinct characteristics compared to other pseudouridine synthases in several key aspects. Unlike most other Ψ synthases that target conserved sequences, truA can modify multiple tRNAs (approximately 17 different tRNAs in E. coli) with highly divergent sequences in the anticodon stem loop region . Additionally, truA possesses remarkable regional specificity, being able to modify nucleotides that are positioned as far as 15 Å apart using a single active site . For instance, in tRNA^leu2, truA can modify uridines at both positions 38 and 40. This "promiscuity" in substrate selection sets truA apart from other pseudouridine synthases like TruB, which modifies only U55 in the conserved T-stem loop of tRNAs .

What is the molecular structure of recombinant E. coli O127:H6 truA?

The recombinant E. coli O127:H6 truA protein consists of 375 amino acids, as indicated by its protein sequence . Based on structural studies of E. coli truA, the enzyme contains a catalytic domain with the active site responsible for pseudouridine formation. Crystal structures have revealed that truA forms complexes with different tRNAs, providing insights into its binding mechanism . The protein sequence of recombinant truA from E. coli O127:H6 is:

MQCALYDAGRCRSCQWITQPIPEQLSAKTADLKNLLADFPVEEWCAPVSGPEQGFRNKAKMVVSGSVEKPLLGMLHRDGTPEDLCDCPLYPASFAPVFAALKPFIARAGLTPYNVARKRGELKYILLTESQSDGGMMLRFVLRSDTKLAQLRKALPWLQEQLPQLKVITVNIQPVHMAIMEGETEIYLTEQQALAERFNDVPLWIRPQSFFQTNPAVASQLYATARDWVRQLPVKHMWDLFCGVGGFGLHCATPDMQLTGIEIAPEAIACAKQSAAELGLTRLQFQALDSTQFASAQGEVPELVLVNPPRRGIGKPLCDYLSTMAPRFIIYSSCNAQTMAKDIRELPGYRIERVQLFDMFPHTAHYEVLTLLVKQ

This sequence information is essential for researchers designing experiments involving protein expression, purification, and functional characterization.

How should researchers design experiments to study truA function in vitro?

To effectively study truA function in vitro, researchers should implement a true experimental design with appropriate controls. Begin by expressing and purifying recombinant truA protein from E. coli O127:H6 using standard molecular cloning techniques. Design your experimental approach with the following considerations:

• Random assignment and control groups: Establish control reactions without truA enzyme alongside experimental reactions to isolate the effect of truA activity . This approach reduces potential biases and ensures that observed changes in tRNA modification are attributable to truA activity.

• Substrate preparation: Prepare various tRNA substrates, particularly focusing on those with uridines at positions 38-40 in the anticodon stem loop. Include tRNAs with different sequence contexts around these positions to evaluate substrate specificity.

• Reaction conditions optimization: Systematically test different buffer compositions, pH values, temperatures, and incubation times to determine optimal conditions for truA activity.

• Detection methods: Employ techniques such as high-performance liquid chromatography (HPLC), mass spectrometry, or radioisotope labeling to detect and quantify pseudouridine formation.

• Kinetic analysis: Measure reaction rates with varying substrate concentrations to determine kinetic parameters such as Km and Vmax, providing insights into truA's efficiency and substrate preference.

This methodological approach will provide reliable and reproducible results when investigating truA function in vitro .

What are the optimal conditions for expressing and purifying recombinant E. coli O127:H6 truA?

For optimal expression and purification of recombinant E. coli O127:H6 truA, researchers should consider the following methodological approach:

Expression System:

• Use an E. coli expression system with a strong inducible promoter (such as T7)

• Transform the expression vector containing the truA gene (Uniprot ID: B7UMV1) into a suitable E. coli strain like BL21(DE3)

• Culture cells in rich media (LB or 2XYT) supplemented with appropriate antibiotics

• Induce protein expression at mid-log phase (OD600 ~0.6-0.8) with IPTG

• Optimize induction temperature (typically 16-30°C) and duration (4-24 hours) to maximize soluble protein yield

Purification Protocol:

• Harvest cells by centrifugation and resuspend in a Tris-based buffer with protease inhibitors

• Lyse cells using sonication or high-pressure homogenization

• Clarify lysate by centrifugation at high speed (≥20,000 × g)

• Employ affinity chromatography (if using a tagged construct) or ion exchange chromatography as the initial purification step

• Further purify using size exclusion chromatography to achieve high purity (>85% as determined by SDS-PAGE)

• Store the purified protein in a Tris-based buffer with 50% glycerol at -20°C or -80°C to maintain stability

This systematic approach will yield high-quality recombinant truA protein suitable for functional and structural studies.

How can researchers accurately identify and quantify pseudouridine modifications introduced by truA?

Accurately identifying and quantifying pseudouridine modifications introduced by truA requires a multi-faceted analytical approach. Researchers should consider implementing the following methodological strategies:

• Chemical derivatization methods: Utilize N-cyclohexyl-N'-(2-morpholinoethyl)carbodiimide metho-p-toluenesulfonate (CMC) treatment followed by alkaline hydrolysis, which specifically labels pseudouridines and allows their detection through reverse transcription stops.

• Mass spectrometry approaches: Implement liquid chromatography-tandem mass spectrometry (LC-MS/MS) to precisely identify and quantify pseudouridines. This technique can distinguish between unmodified uridines and pseudouridines based on their mass difference and fragmentation patterns.

• Next-generation sequencing-based methods: Apply Ψ-seq or similar techniques that combine CMC treatment with high-throughput sequencing to map pseudouridines across multiple tRNA substrates simultaneously.

• Site-specific detection: For comparing multiple experimental conditions, develop tRNA-specific primers that target regions containing positions 38, 39, and 40 to identify the exact location and frequency of modifications.

• Quantitative analysis: Normalize pseudouridine levels against total tRNA or against specific invariant modifications to ensure accurate quantification across different samples and experimental conditions.

This comprehensive approach allows for reliable identification and precise quantification of truA-mediated pseudouridine modifications, essential for understanding the enzyme's activity and specificity .

How does truA select its target sites in tRNAs with such diverse sequences?

The mechanism by which truA selects its target sites in tRNAs with diverse sequences has been elucidated through crystallographic studies and functional assays. Unlike other pseudouridine synthases that recognize specific sequence motifs, truA employs a unique strategy based on structural recognition:

This sophisticated target selection mechanism represents an evolutionary adaptation allowing truA to modify multiple tRNAs with divergent sequences while maintaining specificity for the targeted nucleotides in the ASL region .

What is the catalytic mechanism of truA and how does it differ from other pseudouridine synthases?

The catalytic mechanism of truA involves several distinctive steps that differentiate it from other pseudouridine synthases:

This mechanism represents an elegant solution for RNA modification that balances specificity with versatility, allowing truA to modify multiple tRNAs with diverse sequences while maintaining positional accuracy.

How does pseudouridylation by truA affect tRNA stability and function?

Pseudouridylation by truA significantly impacts tRNA stability and function through several mechanistic pathways:

The modifications introduced by truA are therefore essential for maintaining the delicate structural balance required for optimal tRNA function in protein synthesis, highlighting the biological significance of this enzyme in bacterial physiology.

How can truA be used as a tool to study RNA structure-function relationships?

Recombinant E. coli O127:H6 truA offers several sophisticated applications for investigating RNA structure-function relationships:

• Controlled RNA modification: By manipulating truA activity in vitro, researchers can introduce pseudouridines at specific positions to create defined tRNA variants. This allows for the systematic study of how these modifications influence tRNA folding, stability, and function.

• Structure probing: The enzymatic activity of truA can serve as a probe for tRNA conformational states. Since truA recognizes and modifies flexible regions in the anticodon stem loop, its activity pattern can reveal information about RNA structural dynamics that may not be apparent from static structural analyses.

• Comparative studies: Researchers can design experiments comparing truA from pathogenic E. coli O127:H6 with that from non-pathogenic strains to investigate potential differences in substrate specificity or enzymatic efficiency. This approach requires rigorous experimental design with appropriate controls to ensure valid comparisons .

• Synthetic biology applications: Engineered variants of truA with altered specificity can be developed to introduce pseudouridines at novel positions, creating custom-modified RNAs with potentially enhanced or altered functions for biotechnological applications.

• RNA-protein interaction studies: The truA-tRNA complex serves as an excellent model system for studying the molecular basis of enzyme-RNA recognition and specificity, providing insights into the principles governing RNA-protein interactions.

These advanced applications highlight how recombinant truA can be leveraged as a sophisticated tool for exploring the complex relationship between RNA structure and function in biological systems .

What approaches can be used to analyze contradictory results in truA research?

When confronted with contradictory results in truA research, investigators should implement a systematic analytical approach:

• Experimental design evaluation: Carefully examine the experimental designs of contradictory studies. Small differences in methodology can significantly impact outcomes. True experimental designs with random assignment and proper controls are essential for reliable results .

• Variable identification and control: Create a comprehensive table comparing all experimental variables between contradictory studies, including:

• Replication with methodological triangulation: Reproduce the contradictory results using multiple independent methodologies to determine whether the contradictions are methodological artifacts or represent genuine biological complexity .

• Meta-analysis approach: When possible, perform a systematic meta-analysis of all available data, weighting results based on methodological rigor and sample size to identify consistent patterns or context-dependent effects.

• Computational modeling: Develop computational models to test whether contradictory results might represent different aspects of a more complex underlying mechanism that reconciles apparently conflicting observations.

This structured approach acknowledges that contradictory results may reflect genuine biological complexity rather than experimental error, providing a pathway to deeper understanding of truA function .

How can researchers investigate the relationship between truA activity and bacterial pathogenicity in E. coli O127:H6?

Investigating the relationship between truA activity and pathogenicity in E. coli O127:H6 requires a multidisciplinary approach combining molecular genetics, functional assays, and infection models:

• Gene knockout and complementation studies: Create truA knockout strains of E. coli O127:H6 and complement with wild-type or mutant truA variants. This experimental design allows for direct assessment of how truA contributes to bacterial fitness and virulence .

• Transcriptome and proteome analysis: Compare the transcriptome and proteome profiles of wild-type and truA-deficient strains under various conditions, particularly those mimicking the host environment. This approach can reveal how truA-mediated tRNA modifications influence gene expression patterns related to virulence.

• Translation fidelity assessment: Develop reporter systems to measure the accuracy of protein synthesis in the presence and absence of functional truA. This can determine whether truA-mediated modifications affect the translation of specific virulence factors.

• Host-pathogen interaction models: Utilize cell culture models with intestinal epithelial cells to assess the impact of truA on adhesion, invasion, and host cell response. Progress to more complex models such as organoids or animal models to evaluate pathogenicity in vivo.

• Comparative analysis across strains: Extend the investigation to compare truA function between enteropathogenic E. coli O127:H6 and other E. coli pathotypes to identify potential strain-specific effects .

This comprehensive research approach can elucidate whether truA-mediated tRNA modifications contribute to the pathogenicity of E. coli O127:H6, potentially revealing new targets for antimicrobial development or diagnostic tools.

What are the emerging technologies that could advance truA research?

Several cutting-edge technologies are poised to significantly advance research on recombinant E. coli O127:H6 truA:

• Cryo-electron microscopy (Cryo-EM): This technique can capture truA-tRNA complexes in different conformational states at near-atomic resolution, providing dynamic structural insights that complement existing crystallographic data .

• Single-molecule FRET (smFRET): This approach allows real-time observation of truA-tRNA interactions and conformational changes during the modification process, offering unprecedented insights into the kinetics and mechanism of pseudouridylation.

• Nanopore direct RNA sequencing: This technology enables direct detection of modified nucleotides in native RNA without reverse transcription or amplification, potentially allowing for more accurate mapping of pseudouridines in complex tRNA populations.

• CRISPR-Cas systems for RNA modification: Engineered CRISPR-Cas systems can be developed to target specific RNA sequences for modification, potentially allowing for programmable pseudouridylation in living cells.

• Artificial intelligence and machine learning: These computational approaches can analyze large datasets of tRNA sequences and structures to identify patterns in truA substrate recognition that may not be apparent through conventional analysis.

• High-throughput functional assays: Development of scalable assays to systematically test truA activity across hundreds of substrate variants simultaneously will accelerate our understanding of substrate specificity determinants.

These emerging technologies, when combined with rigorous experimental design principles, have the potential to resolve existing questions and open new avenues of investigation in truA research .

How might a deeper understanding of truA contribute to antibacterial drug development?

A comprehensive understanding of truA function and mechanism could significantly impact antibacterial drug development through several strategic approaches:

• Novel drug target identification: As a conserved enzyme essential for optimal translation, truA represents a potential antibacterial target. The unique catalytic mechanism and substrate recognition features of truA could be exploited to develop selective inhibitors that disrupt bacterial protein synthesis without affecting human pseudouridine synthases.

• Pathogen-specific targeting: Comparative analysis of truA from pathogenic E. coli O127:H6 versus commensal strains could reveal structural or functional differences that enable selective targeting of pathogenic bacteria, minimizing disruption to beneficial gut microbiota .

• Structure-based drug design: The crystal structures of truA in complex with tRNA substrates provide valuable templates for rational design of small-molecule inhibitors that specifically bind to the active site or disrupt critical protein-RNA interactions .

• Combination therapy strategies: Inhibitors targeting truA could be developed as sensitizing agents that enhance the efficacy of existing antibiotics by compromising translational fidelity and adaptive responses in bacteria.

• Resistance mechanism prediction: Understanding the molecular details of truA function allows researchers to predict potential resistance mechanisms, enabling proactive development of second-generation inhibitors that maintain efficacy despite anticipated bacterial adaptations.

This approach to antibacterial drug development, grounded in fundamental understanding of bacterial RNA modification enzymes, offers promising avenues for addressing the growing challenge of antimicrobial resistance, particularly for infections caused by pathogenic E. coli strains .