Recombinant Shewanella baltica tRNA pseudouridine synthase A (truA)

What is tRNA pseudouridine synthase A (truA) and what is its function in Shewanella baltica?

TruA is a highly conserved enzyme that catalyzes the conversion of specific uridines to pseudouridines in the anticodon stem loop (ASL) of tRNAs. In Shewanella baltica, as in other bacteria, truA specifically targets positions 38, 39, and/or 40 of tRNAs with highly divergent sequences and structures . This modification plays a crucial role in maintaining translational accuracy and efficiency by increasing the thermal stability of the ASL, which affects anticodon-codon interactions and tRNA conformational changes during translation .

Given that Shewanella baltica is a cold-adapted marine bacterium capable of growth at 4°C but not at 37°C , its truA likely possesses unique characteristics that enable efficient function at lower temperatures, contributing to the organism's adaptation to cold marine environments.

How does the structure of truA contribute to its substrate recognition?

TruA functions as a homodimer, with each monomer comprising distinct N- and C-terminal domains that form an active site cleft containing the universally conserved catalytic Asp60 . This dimeric structure is critical for function as tRNA binds across both subunits. The ASL binds in the cleft between the N- and C-terminal domains, positioning nucleotides 38-40 near the catalytic aspartate residue .

What makes truA remarkable is its structural basis for "substrate promiscuity." Unlike other pseudouridine synthases that recognize specific conserved sequences, truA recognizes the common shape and electrostatic properties of tRNAs, primarily interacting with the elbow where the D and T loops join together and the D-stem backbone . This allows truA to modify multiple tRNAs with divergent sequences, highlighting its unique regional specificity rather than sequence specificity.

What experimental approaches are used to characterize the cold adaptation of Shewanella baltica truA?

To investigate the cold adaptation features of Shewanella baltica truA, researchers should implement the following methodological approaches:

• Comparative thermal activity profiling: Measure enzymatic activity across a temperature range (0-40°C) and compare with mesophilic homologs to determine temperature optima and activity range.

• Structural flexibility analysis: Employ hydrogen-deuterium exchange mass spectrometry (HDX-MS) to assess protein dynamics at different temperatures, particularly focusing on active site regions.

• Thermostability measurements: Use differential scanning fluorimetry (DSF) or circular dichroism (CD) to determine melting temperatures and thermal unfolding profiles.

• Kinetic parameter determination: Calculate Km, kcat, and activation energy (Ea) values at multiple temperatures to establish how cold adaptation affects catalytic efficiency.

These methods can reveal how Shewanella baltica truA has evolved structural modifications to maintain catalytic efficiency at temperatures corresponding to its marine habitat .

What expression systems are optimal for producing recombinant Shewanella baltica truA?

Selecting the appropriate expression system is critical for obtaining functionally active Shewanella baltica truA. Based on the cold-adapted nature of this organism , the following methodological considerations are recommended:

• Host selection: E. coli Arctic Express or BL21(DE3) strains are preferred hosts, as they have been successfully used for other truA proteins . Arctic Express co-expresses cold-adapted chaperonins that may facilitate proper folding of Shewanella proteins.

• Expression vector optimization: Design a construct with codon optimization for E. coli while retaining Shewanella baltica rare codons in critical regions. Include a removable affinity tag (His6 or SUMO) at the N-terminus to minimize interference with dimerization.

• Induction conditions: Use low-temperature induction (12-15°C) for 16-24 hours with reduced IPTG concentration (0.1-0.3 mM) to promote proper folding of this cold-adapted enzyme.

• Expression validation protocol:

  • Monitor expression by SDS-PAGE and Western blotting

  • Perform small-scale activity assays to confirm functionality

  • Analyze soluble versus insoluble fractions to optimize conditions

For a psychrophilic enzyme like Shewanella baltica truA, expression at higher temperatures may lead to misfolding and inclusion body formation, necessitating these cold-adapted expression strategies.

What purification challenges are specific to Shewanella baltica truA and how can they be addressed?

Purifying Shewanella baltica truA presents unique challenges related to its cold adaptation and RNA-binding properties. A comprehensive purification strategy should include:

• Low-temperature processing: Maintain all purification steps at 4°C to preserve the native structure of this cold-adapted enzyme .

• Nucleic acid contamination removal: Implement a sequential approach:

  • High-salt washes (500-700 mM NaCl) during initial chromatography steps

  • Treatment with benzonase nuclease during lysis

  • Polyethyleneimine precipitation (0.15-0.2%) to remove nucleic acids

  • Ion exchange chromatography with shallow gradients

• Dimeric state preservation: Given that truA functions as a homodimer , include stabilizing agents in all buffers:

  • 5-10% glycerol to prevent subunit dissociation

  • 1-2 mM DTT to maintain reduced cysteines

  • Moderate salt concentration (150-200 mM) to preserve ionic interactions

• Quality control assessments:

  • Size exclusion chromatography to confirm dimeric state

  • Dynamic light scattering to evaluate homogeneity

  • Activity assays at each purification stage to track specific activity

This stepwise approach addresses the specific challenges of maintaining cold-adapted enzyme stability while achieving high purity.

How can I assess and optimize the activity of recombinant Shewanella baltica truA?

Developing robust activity assays for Shewanella baltica truA requires consideration of its cold adaptation and multiple target sites. A comprehensive activity assessment strategy includes:

• Pseudouridylation detection methods:

  • CMC-primer extension assay: Treat modified tRNA with N-cyclohexyl-N'-(2-morpholinoethyl)carbodiimide metho-p-toluenesulfonate (CMC), which specifically reacts with pseudouridine and blocks reverse transcription

  • HPLC analysis of nucleoside composition after complete enzymatic digestion of tRNA

  • Mass spectrometry to detect pseudouridine-specific fragmentation patterns

• Substrate selection considerations:

  • Use multiple tRNAs with uridines at positions 38, 39, and/or 40 to assess site preference

  • Include both homologous (Shewanella) and heterologous (E. coli) tRNAs to evaluate substrate specificity

  • Prepare in vitro transcribed tRNAs lacking any modifications for controlled experiments

• Reaction condition optimization:

  • Temperature range: Test activity at 4-25°C to determine temperature optimum

  • Buffer composition: Evaluate various pH values (6.5-8.0) and salt concentrations (50-300 mM)

  • Divalent cations: Assess requirements for Mg²⁺, Mn²⁺, or other cofactors

  • Reducing environment: Optimize DTT or β-mercaptoethanol concentration

• Kinetic parameter determination:

  • Measure initial rates at varying substrate concentrations

  • Determine Km, kcat, and catalytic efficiency (kcat/Km) at different temperatures

  • Compare with E. coli truA to quantify cold adaptation effects

This multifaceted approach provides comprehensive characterization of the recombinant enzyme's activity and specificity profile.

What structural techniques are most informative for studying Shewanella baltica truA-tRNA interactions?

Investigating the structural basis of Shewanella baltica truA-tRNA interactions requires complementary techniques that capture different aspects of this dynamic complex:

Each method provides complementary information, collectively revealing how this cold-adapted enzyme recognizes and modifies its tRNA substrates.

How does Shewanella baltica truA achieve site specificity for positions 38-40 in tRNAs?

The mechanism by which truA achieves its unique regional specificity for positions 38-40 in tRNAs with diverse sequences represents a fascinating aspect of RNA-protein recognition. Based on structural studies of E. coli truA , the following model explains this specificity:

• Initial recognition: TruA recognizes the global architecture of tRNA rather than specific sequences, binding across both subunits of the enzyme dimer . The elbow region where the D and T loops join and the D-stem backbone serve as primary recognition elements .

• Multistep binding process: The crystal structures reveal three distinct conformational states during binding :

  • Initial complex: tRNA body docks distal to the active site

  • Intermediate state: The anticodon stem loop (ASL) bends toward the active site cleft

  • Reactive conformation: Target base flips out and positions in the active site

• Base-flipping mechanism: The enzyme exploits the intrinsic flexibility of the ASL to promote base-flipping of the target uridines . This is evidenced by crystal structures showing a flipped-out G39 in the active site, suggesting any base at position 39 can be accommodated .

• Regional targeting: The positioning of the ASL relative to the active site creates a "recognition window" where nucleotides 38-40 can sequentially access the catalytic center through conformational adjustments.

For Shewanella baltica truA, cold adaptation may enhance the flexibility of key structural elements involved in this process, potentially modifying the kinetics of base-flipping or the conformational landscape of the enzyme-tRNA complex.

What is the catalytic mechanism of truA and how might cold adaptation modify it?

The catalytic mechanism of truA involves several discrete steps that convert uridine to pseudouridine in tRNA. Based on structural and biochemical studies of E. coli truA , the reaction proceeds as follows:

• Base flipping: The target uridine is flipped out of the ASL and positioned in the active site, as observed in crystal structures .

• Nucleophilic attack: The conserved catalytic Asp60 attacks C6 of uridine, forming a covalent enzyme-RNA intermediate .

• Glycosidic bond cleavage: The N1-C1' glycosidic bond is broken, releasing the uracil base while maintaining the covalent link to the enzyme.

• Base rotation: The uracil moiety rotates 180° within the active site.

• Re-attachment: The C5 position of uracil attacks the C1' of ribose, forming the characteristic C-C glycosidic bond of pseudouridine.

• Product release: The modified tRNA dissociates from the enzyme.

For Shewanella baltica truA, cold adaptation likely modifies this mechanism in several ways:

These adaptations would maintain catalytic efficiency at temperatures relevant to Shewanella baltica's natural marine environment (4°C) , potentially through reduced enthalpic barriers that compensate for decreased thermal energy.

How can comparative studies of Shewanella baltica truA inform our understanding of enzyme cold adaptation?

Shewanella baltica truA represents an excellent model system for investigating fundamental principles of enzyme cold adaptation due to its essential function and the wealth of structural and mechanistic data available from mesophilic homologs . A comprehensive research strategy would include:

• Comparative structural biology:

  • Solve high-resolution structures of Shewanella baltica truA and compare with E. coli truA

  • Analyze differences in surface charge distribution, internal packing, and loop flexibility

  • Identify structural modifications that facilitate function at low temperatures

• Comparative enzymology:

  • Measure temperature-dependence of kinetic parameters (kcat, Km, kcat/Km)

  • Determine comparative activation energies and thermodynamic parameters

  • Analyze the temperature-dependence of protein dynamics using HDX-MS or NMR

• Directed evolution and domain-swapping experiments:

  • Create chimeric enzymes combining domains from psychrophilic and mesophilic truA

  • Perform site-directed mutagenesis to introduce/remove cold-adaptive features

  • Assess the minimum modifications needed to convert between temperature adaptations

• Computational simulations:

  • Perform molecular dynamics at different temperatures to identify regions with differential flexibility

  • Calculate free energy landscapes for catalytic steps under varying temperature conditions

  • Predict key residues involved in cold adaptation through statistical analysis of homologous sequences

These approaches would reveal molecular mechanisms underlying cold adaptation that could be applied to other enzyme systems and potentially inform protein engineering strategies for cold-active biocatalysts.

What role might Shewanella baltica truA play in environmental adaptation of the organism?

The functional significance of truA in Shewanella baltica's adaptation to cold marine environments represents an intriguing aspect of environmental microbiology and RNA biology:

• Translational efficiency at low temperatures:

  • Pseudouridylation in the anticodon stem loop (ASL) increases its thermal stability , which may be particularly important in cold environments where RNA structure is more rigid

  • This modification could maintain optimal codon-anticodon interactions at low temperatures where weak interactions are further destabilized

  • Enhanced translational accuracy could compensate for slower protein synthesis rates in cold conditions

• Regulatory mechanisms during temperature shifts:

  • TruA activity may be modulated by temperature, creating a temperature-sensitive translation control mechanism

  • Differential modification of certain tRNAs could regulate the expression of cold-responsive genes

  • The ratio of modified to unmodified tRNAs could serve as a temperature-sensing mechanism

• Experimental approaches to test these hypotheses:

  • Compare pseudouridylation levels in tRNAs isolated from Shewanella baltica grown at different temperatures

  • Perform ribosome profiling to identify changes in translation efficiency correlated with tRNA modification status

  • Create truA deletion or catalytically inactive mutants and assess their growth at different temperatures

• Ecological significance:

  • As Shewanella baltica is found in the Baltic Sea and plays a role in fish spoilage , its cold adaptation mechanisms have both ecological and economic relevance

  • Understanding these adaptations could provide insights into microbial community dynamics in seasonally variable marine environments

This research direction connects molecular enzymology with ecological adaptation and could reveal new principles of RNA-based temperature adaptation in psychrophilic organisms.

How can recombinant Shewanella baltica truA be used as a tool in RNA modification studies?

Recombinant Shewanella baltica truA offers unique properties that make it valuable for various applications in RNA modification research:

• Cold-active RNA modification tool:

  • Enables pseudouridylation reactions at lower temperatures (4-15°C) where RNA secondary structures remain intact

  • Potentially reduces unwanted RNA degradation during long modification reactions

  • May offer different substrate specificity compared to mesophilic enzymes

• Site-specific RNA labeling applications:

  • TruA's ability to target positions 38-40 in diverse tRNAs can be exploited for position-specific RNA labeling

  • Can be combined with nucleotide analogs that contain chemical handles for downstream conjugation

  • Methodology development:

    • Incorporate 5-azauridine at target positions in synthetic RNA

    • Use truA to convert to 5-azapseudouridine

    • Perform click chemistry to attach fluorophores or affinity tags

• Structural biology applications:

  • Generate uniformly pseudouridylated tRNAs for structural studies

  • Compare structures of modified vs. unmodified RNAs under various temperature conditions

  • Investigate the impact of pseudouridylation on RNA-protein interactions

• Biotechnological applications:

  • Engineer truA variants with altered specificity through directed evolution

  • Develop cold-active RNA modification systems for temperature-sensitive RNA substrates

  • Create bifunctional enzymes combining truA activity with other RNA modifying activities

These applications leverage the unique properties of this cold-adapted enzyme to expand the toolkit for RNA manipulation and analysis.

What strategies can overcome common challenges in Shewanella baltica truA expression and purification?

Recombinant expression of psychrophilic enzymes like Shewanella baltica truA presents specific challenges that require methodical troubleshooting approaches:

• Low expression yield challenges:

  • Problem: Cold-temperature induction reduces expression levels

  • Solution: Optimize by testing multiple E. coli strains (Rosetta, Arctic Express, BL21-AI)

  • Methodology: Implement auto-induction media specifically designed for low-temperature expression

  • Validation: Compare protein yields using Western blot and activity assays

• Protein solubility issues:

  • Problem: Improper folding at expression temperatures above Shewanella's growth range

  • Solution: Test solubility enhancement tags (SUMO, MBP, TrxA) with various induction temperatures

  • Methodology: Perform systematic expression screening with factorial design:

    • Temperature range: 8°C, 12°C, 15°C, 18°C

    • Induction time: 16h, 24h, 36h, 48h

    • IPTG concentration: 0.1mM, 0.25mM, 0.5mM

  • Analysis: Quantify soluble fraction percentage using densitometry

• Nucleic acid contamination:

  • Problem: truA's natural affinity for RNA leads to co-purification with cellular RNA

  • Solution: Implement a multi-step nucleic acid removal strategy

  • Methodology:

    • Pre-treat lysate with benzonase (25U/mL) for 30 minutes at 4°C

    • Include stepwise salt washes (200mM, 500mM, 750mM NaCl)

    • Apply subtractive ion-exchange chromatography

  • Quality control: Monitor A260/A280 ratio (<0.7 indicates low nucleic acid contamination)

• Enzyme instability during purification:

  • Problem: Activity loss during purification steps

  • Solution: Optimize buffer composition for cold-adapted enzyme stability

  • Methodology: Systematic buffer screening with thermal shift assay:

    • pH range: 6.5-8.0

    • Salt type and concentration

    • Stabilizing additives (glycerol, sucrose, arginine)

  • Validation: Measure specific activity at each purification step

These methodical approaches address the specific challenges associated with this psychrophilic enzyme while maintaining its native properties.

How can I develop reliable activity assays for Shewanella baltica truA?

Developing robust and quantitative activity assays for Shewanella baltica truA requires consideration of its cold adaptation and site-specific activity. A comprehensive assay development strategy includes:

• Tritium release assay optimization:

  • Principle: [³H]-labeled uridine in tRNA substrate releases tritium during pseudouridylation

  • Methodology:

    • Prepare [5-³H]-UTP labeled tRNA substrates through in vitro transcription

    • Incubate with truA at 4-25°C in optimized reaction buffer

    • Separate released [³H] using activated charcoal adsorption

    • Quantify tritium in supernatant by scintillation counting

  • Controls: Include heat-inactivated enzyme and catalytically inactive D60A mutant

• CMC-primer extension assay refinement:

  • Principle: CMC specifically modifies pseudouridine and blocks reverse transcription

  • Methodology:

    • Optimize CMC reaction conditions for cold-temperature application

    • Develop fluorescent primer labeling for quantitative detection

    • Use capillary electrophoresis for high-resolution band separation

  • Quantification: Implement standard curves with synthetic pseudouridylated RNA

• Mass spectrometry-based quantification:

  • Principle: Precise detection of pseudouridine versus uridine in digested RNA

  • Methodology:

    • Optimize enzymatic digestion to nucleosides

    • Develop LC-MS/MS method with appropriate separation column

    • Implement multiple reaction monitoring (MRM) for sensitivity

  • Analysis: Calculate modification percentage based on pseudouridine/uridine ratio

• Real-time fluorescence monitoring:

  • Principle: Base-flipping during catalysis affects local environment of fluorescent nucleotide analogs

  • Methodology:

    • Incorporate 2-aminopurine adjacent to target uridines

    • Monitor fluorescence changes during reaction progress

    • Optimize excitation/emission parameters for low-temperature reactions

  • Applications: Enables continuous kinetic measurements at various temperatures

This multifaceted approach provides complementary methods for comprehensive characterization of Shewanella baltica truA activity under various experimental conditions.