Recombinant Thermoproteus neutrophilus tRNA pseudouridine synthase A (truA)

What is Thermoproteus neutrophilus tRNA pseudouridine synthase A and what is its function?

Thermoproteus neutrophilus tRNA pseudouridine synthase A (truA) belongs to the pseudouridine synthase family that catalyzes the isomerization of uridine to pseudouridine at specific positions in tRNA molecules. This post-transcriptional modification is crucial for proper tRNA function during protein synthesis in this hyperthermophilic archaeon that grows at 85°C under strictly anaerobic conditions .

The enzyme specifically targets tRNA molecules, where the conversion of uridine to pseudouridine helps stabilize RNA structure through additional hydrogen bonding capabilities. This modification is particularly important in thermophiles like T. neutrophilus, where increased structural stability is required for proper tRNA function at high temperatures .

How does T. neutrophilus differ from other archaeal species in terms of metabolism and carbon fixation?

T. neutrophilus is a strictly anaerobic hyperthermophilic archaeon that grows autotrophically by reducing sulfur with hydrogen at 85°C and neutral pH. Unlike many other Crenarchaeota that use the 3-hydroxypropionate/4-hydroxybutyrate cycle for carbon fixation, T. neutrophilus employs the dicarboxylate/4-hydroxybutyrate (DC/HB) cycle .

While the reductive citric acid cycle was initially proposed as the carbon fixation pathway in T. neutrophilus, subsequent genomic analysis revealed the absence of a key enzyme (ATP-citrate lyase) and the presence of 4-hydroxybutyryl-CoA dehydratase, indicating that the DC/HB cycle is the actual pathway used . This cycle involves seven steps to convert succinyl-CoA to two molecules of acetyl-CoA, with several enzymes specific to this pathway having been identified and characterized .

What are the key structural features that distinguish archaeal pseudouridine synthases from bacterial counterparts?

Archaeal pseudouridine synthases exhibit several distinctive structural features compared to their bacterial counterparts:

• Domain architecture: Many archaeal pseudouridine synthases possess unique domains, such as the THUMP domain found in Pus10, which is involved in substrate recognition and binding .

• Catalytic motif: While both archaeal and bacterial enzymes contain a catalytic domain with the conserved "GRLD" motif, archaeal variants often show modifications to this motif that maintain function under extreme conditions .

• Thermostability adaptations: T. neutrophilus enzymes contain additional structural elements that enhance stability at high temperatures (85°C), including increased hydrophobic interactions, additional salt bridges, and compact folding .

• Substrate recognition loops: Archaeal pseudouridine synthases contain specialized loops, such as the FFL and thumb-loop in Pus10, which contribute to position-specific modifications and substrate recognition .

Multiple sequence alignment of Pyrobaculum arsenaticum, P. islandicum and Thermoproteus neutrophilus shows modifications in all structural features for Pus10 compared to bacterial enzymes, highlighting their evolutionary divergence while maintaining similar catalytic functions .

What are the optimal conditions for expressing and purifying recombinant T. neutrophilus tRNA pseudouridine synthase A?

Based on protocols used for similar enzymes from thermophilic archaea, the following conditions are recommended:

Expression System:

• E. coli expression using vectors like pET16b (N-terminal His10 tag) or pT7-7

• Transformation into E. coli DH5α for cloning and BL21(DE3) for expression

• Verification of insert sequence prior to expression

Expression Protocol:

• Culture in LB medium with appropriate antibiotics

• Induction with IPTG (0.5-1.0 mM) at OD600 of 0.6-0.8

• Expression at 30°C for 4-6 hours or 18°C overnight to enhance solubility

Purification Steps:

• Cell lysis via sonication in buffer containing protease inhibitors

• Heat treatment (65-75°C for 15-20 minutes) to denature most E. coli proteins

• Immobilized metal affinity chromatography using Ni-NTA resin

• Size exclusion chromatography for final purification

• Storage in Tris-based buffer with 50% glycerol at -20°C/-80°C

The recombinant protein should achieve greater than 85% purity as determined by SDS-PAGE. For long-term storage, avoid repeated freeze-thaw cycles. The shelf life is approximately 6 months at -20°C/-80°C in liquid form and 12 months in lyophilized form .

What PCR conditions should be used to amplify the T. neutrophilus tRNA pseudouridine synthase gene?

Based on successful amplification of other genes from T. neutrophilus, the following PCR conditions are recommended:

Enzyme Mixture:

• Use a combination of Taq and Pfu polymerases for both efficiency and fidelity

• Recommended ratio: 10:1 Taq:Pfu

Cycling Parameters:

• Initial denaturation: 94°C for 3 minutes

• 35 cycles consisting of:

  • Denaturation: 94°C for 30 seconds

  • Annealing: 60-65°C for 30 seconds (optimize based on primer design)

  • Extension: 72°C for 90-105 seconds (for genes ~1kb in length)

• Final extension: 72°C for 10 minutes

Primer Design:

• Include appropriate restriction sites for subsequent cloning

• Add 3-6 nucleotide overhangs before restriction sites

• Design primers with GC content between 40-60%

Reaction Components:

• Template DNA: 50-100 ng of T. neutrophilus genomic DNA

• Primers: 0.5 μM each

• dNTPs: 200 μM each

• MgCl₂: 1.5-2.0 mM

• Buffer: Use buffer provided with polymerase

• DMSO: Consider adding 3-5% for GC-rich templates

After amplification, purify the PCR product using a QIAquick PCR purification kit before restriction enzyme digestion and ligation into the expression vector .

How can enzyme activity assays be optimized for thermostable tRNA pseudouridine synthases?

Optimizing enzyme activity assays for thermostable tRNA pseudouridine synthases requires specific considerations for high-temperature reactions:

Substrate Preparation:

• Generate in vitro transcribed tRNA substrates using T7 RNA polymerase

• Purify tRNA by anion exchange chromatography to ensure homogeneity

• For radioactive assays, incorporate labeled UTP during transcription

Reaction Conditions:

• Buffer stability: Use buffers with minimal temperature-dependent pH shifts (e.g., HEPES, phosphate)

• Temperature: Conduct assays at physiologically relevant temperatures (65-85°C for T. neutrophilus)

• Reaction vessels: Use PCR tubes or specialized high-temperature microplates to minimize evaporation

• Include stabilizing agents: Consider adding BSA (0.1 mg/ml) or glycerol (5%) to enhance enzyme stability

Detection Methods:

• Tritium Release Assay:

  • Use [5-³H]-labeled tRNA substrates

  • Measure released tritium as an indication of pseudouridine formation

  • Separate enzyme-bound from free tritium using resin filtration

• TLC-Based Analysis:

  • Digest modified tRNA with nuclease P1

  • Analyze digested products by 2D-TLC

  • Visualize through autoradiography or phosphorimaging

• Spectrophotometric Monitoring:

  • Measure absorbance changes when tRNA binds to the enzyme

  • The search results indicate: "When tRNA Phe is rapidly mixed with buffer instead of TruB, no absorbance change is observed"

• Nearest Neighbor Analysis:

  • Use RNase T2 digestion on radiolabeled tRNA samples

  • Identify modified nucleotides and their sequence context

When analyzing results, plot enzyme activity versus temperature to determine the optimal temperature range, and consider the effects of substrate concentration, buffer composition, and ionic strength on reaction rates.

How does the catalytic mechanism of T. neutrophilus tRNA pseudouridine synthase differ from other pseudouridine synthases?

The catalytic mechanism of T. neutrophilus tRNA pseudouridine synthase involves several distinct features adapted to its thermophilic lifestyle:

Reaction Steps:

• Binding of tRNA substrate with initial conformational changes

• Flipping of target uridine into the catalytic pocket

• Nucleophilic attack on C1' by an active site aspartate

• Formation of a glycal intermediate with cleavage of the N-glycosidic bond

• Rotation of the uracil base

• Re-formation of the C-C glycosidic bond

• Product release

Key Mechanistic Differences:

• Temperature Adaptation: The enzyme maintains catalytic efficiency at much higher temperatures (85°C) than mesophilic counterparts

• Conformational Dynamics: Pre-steady-state kinetic analysis reveals rapid binding events: "Fitting to a one-exponential equation revealed an apparent rate, k, of 12.7 sec⁻¹"

• Substrate Recognition: Unlike bacterial TruB which modifies only position U55, some archaeal enzymes can modify multiple positions (U54, U55)

• Structural Rearrangements: The interaction requires several conformational changes: "Three bases flip out of the TΨC loop into the catalytic pocket" and "partial unfolding of the D and TΨC arm contributes mostly to the absorbance change"

The active site architecture includes the conserved catalytic residues in the "GRLD" motif, though some archaeal enzymes show variations in this motif while maintaining catalytic function through compensatory mechanisms .

What evolutionary relationships exist between T. neutrophilus pseudouridine synthases and those from other domains of life?

Evolutionary analysis of T. neutrophilus pseudouridine synthases reveals complex relationships with enzymes from other domains of life:

Phylogenetic Classification:

• T. neutrophilus pseudouridine synthases belong to the TruA family, one of several distinct families (TruA, TruB, TruD, RluA, RsuA, and Pus10) in the pseudouridine synthase superfamily

• Despite functional similarities, these enzymes have distinct evolutionary origins, with archaeal enzymes often showing unique domain architectures

Conservation Patterns:

• The catalytic domain containing the "GRLD" motif is generally conserved across all domains of life, suggesting an ancient origin predating the divergence of Bacteria and Archaea

• Archaeal-specific features include modifications in structural elements and additional domains like the THUMP domain in Pus10

• Multiple sequence alignment between Pyrobaculum arsenaticum, P. islandicum, and Thermoproteus neutrophilus shows conservation within the Thermoproteales order but divergence from other archaeal lineages

Functional Convergence:

• Archaeal Pus10 can functionally replace bacterial TruB for U55 modification, suggesting convergent evolution of function

• Some archaeal enzymes like TrmY work in conjunction with pseudouridine synthases to produce methylated derivatives (m¹Ψ), representing archaeal-specific RNA modification pathways

This evolutionary diversity explains why "functional predictions based solely on genome interpretation [are] difficult, if not questionable" for many of these enzymes .

How can data inconsistencies in pseudouridine synthase activity measurements be reconciled and analyzed?

When facing inconsistent data in pseudouridine synthase activity measurements, researchers should apply a systematic approach to reconciliation and analysis:

Step 1: Identify Source Variables

• Experimental Conditions: Temperature, pH, buffer composition, salt concentration

• Enzyme Preparation: Expression system, purification method, presence/absence of tags

• Substrate Differences: Natural vs. synthetic tRNA, full-length vs. stem-loop fragments

• Detection Methods: Direct vs. indirect measurement, sensitivity limits

Step 2: Statistical Analysis

• Apply appropriate statistical tests based on data distribution

• For kinetic data, use non-linear regression analysis to determine Km and kcat values

• Consider weighted regression (1/Y² weighting) for enzymatic data

• Compare different kinetic models using Akaike Information Criterion (AIC)

Step 3: Data Visualization and Tabulation
Creating data tables helps identify patterns in inconsistencies. For example:

Step 4: Bridging In Vitro and In Vivo Data
The search results highlight how seemingly contradictory results can be reconciled through careful analysis: "E. coli deleted in the truB gene grew normally on all media tested. It did exhibit a competitive disadvantage in extended co-culture with its wild-type progenitor and a defect in surviving rapid transfers from 37°C to 50°C" . This indicates that:

• Essential vs. beneficial functions should be distinguished

• Stress conditions may reveal phenotypes not apparent under optimal conditions

• Competitive assays may detect subtle fitness differences missed in simple growth assays

Step 5: Validation through Complementary Methods
For conclusive analysis, employ multiple orthogonal methods:

• Compare steady-state and pre-steady-state kinetics

• Supplement biochemical assays with structural studies

• Verify in vitro findings through in vivo mutant complementation

How can T. neutrophilus tRNA pseudouridine synthase be used to study RNA modification in extremophiles?

T. neutrophilus tRNA pseudouridine synthase provides an excellent model system for studying RNA modifications in extremophiles due to several unique characteristics:

Experimental Approaches:

• Comparative Biochemistry: Directly compare enzymatic properties of T. neutrophilus pseudouridine synthase with mesophilic homologs to identify thermoadaptation mechanisms

  • Measure kinetic parameters at various temperatures

  • Determine temperature-dependent conformational changes

  • Map structural elements contributing to thermostability

• Substrate Specificity Profiling:

  • Test the enzyme against various tRNA substrates from different organisms

  • Identify target recognition patterns specific to thermophilic enzymes

  • Create chimeric tRNAs to map recognition elements

• In vitro Evolution:

  • Use directed evolution to identify mutations enhancing thermostability

  • Test activity-stability tradeoffs in engineered variants

  • Apply findings to create hyperstable RNA modification enzymes for biotechnology

• System-Level Analysis:

  • Study the interplay between different RNA modification enzymes in thermophilic archaea

  • Analyze how modifications contribute to RNA stability at high temperatures

  • Compare modification patterns across thermophiles with different growth optima

The unique properties of T. neutrophilus, including its growth at 85°C and strictly anaerobic lifestyle, make its RNA modification enzymes particularly valuable for understanding how RNA structure and function are maintained under extreme conditions .

What structural modeling approaches are most effective for predicting T. neutrophilus pseudouridine synthase interactions with tRNA?

Effective structural modeling of T. neutrophilus pseudouridine synthase-tRNA interactions requires a multi-faceted approach:

Homology Modeling Strategy:

• Template Selection:

  • Identify closest structural homologs in PDB (e.g., TruB-tRNA or Pus10-tRNA complexes)

  • Evaluate sequence identity and structural conservation of binding regions

  • Consider both archaeal and bacterial templates for comprehensive comparison

• Model Building:

  • Generate multiple models using platforms like SWISS-MODEL, MODELLER, or AlphaFold

  • Pay special attention to loop regions involved in tRNA recognition

  • Model the enzyme both in free and substrate-bound conformations

• tRNA Docking:

  • Prepare tRNA structures accounting for known pseudouridylation targets

  • Use both rigid and flexible docking approaches to accommodate conformational changes

  • Focus on the catalytic pocket and known RNA-binding motifs

• Model Validation and Refinement:

  • Molecular dynamics simulations at high temperatures (65-85°C) to mimic physiological conditions

  • Verify that catalytic residues are properly positioned for the isomerization reaction

  • Compare predicted binding interface with experimental data

Critical Features to Model:

• Base-flipping mechanism: "Three bases flip out of the TΨC loop into the catalytic pocket"

• Conformational changes in tRNA: "Partial unfolding of the D and TΨC arm"

• Thermostability elements specific to T. neutrophilus

The most successful models will account for the unique conformational changes observed in both enzyme and tRNA during catalysis, as described in the presteady-state kinetic studies: "The absorbance change is a result of an early event in the interaction of TruB and tRNA which takes place before catalysis" .

How might CRISPR-Cas genome editing be applied to study pseudouridine synthase function in T. neutrophilus?

Applying CRISPR-Cas genome editing to study pseudouridine synthase function in T. neutrophilus requires specialized approaches for this hyperthermophilic archaeon:

Technical Considerations:

• Thermostable CRISPR-Cas Systems:

  • Select Cas9 or Cas12a variants with activity at high temperatures

  • Consider naturally thermostable Cas proteins from thermophilic bacteria/archaea

  • Engineer existing Cas variants for enhanced thermostability

• Guide RNA Design:

  • Use algorithms that account for the high GC content of T. neutrophilus genome

  • Design guide RNAs to target conserved regions of pseudouridine synthase genes

  • Include appropriate archaeal promoters for guide RNA expression

• Delivery Methods:

  • Develop protocols for electroporation or protoplast transformation optimized for T. neutrophilus

  • Consider conjugation-based methods if direct transformation proves challenging

  • Use selectable markers suitable for thermophilic archaea

Experimental Applications:

• Gene Knockout Studies:

  • Create complete gene deletions to assess essentiality under various growth conditions

  • Compare growth at different temperatures to assess temperature-dependent phenotypes

  • Analyze competitive growth with wild-type strains, similar to studies where "E. coli deleted in the truB gene exhibited a competitive disadvantage in extended co-culture with its wild-type progenitor"

• Domain Mutagenesis:

  • Introduce precise mutations in catalytic residues ("GRLD" motif)

  • Modify RNA-binding regions to alter substrate specificity

  • Create chimeric enzymes by swapping domains with other pseudouridine synthases

• Promoter Modifications:

  • Replace native promoters with inducible systems to control expression levels

  • Create reporter fusions to study expression patterns under different conditions

• Multi-Omics Integration:

  • Combine genome editing with transcriptomics and proteomics

  • Analyze global impacts of pseudouridine synthase modifications on translation

  • Identify potential compensatory mechanisms in response to modification defects

This approach would provide unprecedented insights into the in vivo function of these enzymes in their native thermophilic context.

What are common challenges in expressing recombinant T. neutrophilus pseudouridine synthase in E. coli and how can they be addressed?

Researchers frequently encounter specific challenges when expressing thermophilic archaeal proteins like T. neutrophilus pseudouridine synthase in E. coli:

Challenge 1: Protein Misfolding and Inclusion Body Formation

• Solution: Lower induction temperature to 18-25°C

• Solution: Use specialized E. coli strains (Arctic Express, Rosetta) for improved folding

• Solution: Co-express molecular chaperones (GroEL/GroES) to assist folding

• Solution: Add solubility-enhancing fusion tags (SUMO, MBP, TrxA) rather than just His-tags

Challenge 2: Codon Usage Bias

• Solution: Use codon-optimized synthetic genes for E. coli expression

• Solution: Transform into E. coli strains supplying rare tRNAs (Rosetta, CodonPlus)

• Solution: Analyze codon adaptation index (CAI) to identify problematic regions

Challenge 3: Protein Toxicity to Host Cells

• Solution: Use tightly regulated expression systems (T7-lac, arabinose-inducible)

• Solution: Reduce inducer concentration and extend expression time

• Solution: Consider cell-free expression systems for highly toxic proteins

Challenge 4: Low Yield or Activity

• Solution: Verify protein integrity by mass spectrometry

• Solution: Optimize purification protocol to minimize denaturation

• Solution: Include stabilizing agents (glycerol, specific ions) in buffers

• Solution: Test enzyme activity immediately after purification

Challenge 5: RNA Contamination

• Solution: Include RNase treatment during purification

• Solution: Add high salt washes (500-800 mM NaCl) during affinity chromatography

• Solution: Validate RNA-free preparations by measuring A260/A280 ratio

Quality Control Checklist:

• SDS-PAGE to confirm protein size and >85% purity

• Western blot using anti-His antibodies for tagged constructs

• Mass spectrometry to verify intact protein mass

• Circular dichroism to assess secondary structure

• Activity assays at both mesophilic and thermophilic temperatures

Implementing these strategies will help overcome common expression challenges while ensuring the recombinant enzyme retains its native properties.

How should researchers address contradictory data in tRNA modification pathway analysis?

When confronting contradictory data in tRNA modification pathway analysis, researchers should implement a systematic approach:

Step 1: Data Validation and Verification

• Repeat key experiments with appropriate controls

• Perform intra-laboratory validation with different operators

• Consider inter-laboratory validation for highly contradictory results

Step 2: Methodology Assessment

• Experimental Conditions: Compare reaction temperatures, buffers, incubation times

• Enzyme Sources: Evaluate differences between recombinant vs. native enzymes

• Detection Limits: Assess sensitivity of different analytical methods

Step 3: Reconciliation Framework

• Create a data comparison table to visualize contradictions:

Step 4: Hypothesis Testing

• Design experiments specifically targeting contradictions

• Test boundary conditions between contradictory results

• Consider biological relevance of in vitro vs. in vivo findings

Case Study Example:
The search results highlight a specific contradiction regarding carbon fixation pathways in T. neutrophilus: "The reductive citric acid cycle was proposed for Thermoproteus neutrophilus" but "genomic analysis revealed a surprising feature... the presence of a 4-hydroxybutyryl-CoA dehydratase gene without the presence of an ATP-citrate lyase gene... indicating a possible functioning of the dicarboxylate/4-hydroxybutyrate cycle" .

This contradiction was resolved through multiple lines of evidence:

• Activity measurements of key enzymes

• Incorporation studies with labeled substrates

• Genomic analysis identifying pathway-specific genes

• Comparative analysis with related species

What analytical methods are most reliable for confirming successful tRNA pseudouridylation?

Reliable confirmation of successful tRNA pseudouridylation requires complementary analytical approaches:

Method 1: Nucleoside Analysis by LC-MS/MS

• Procedure: Enzymatically digest tRNA to nucleosides, separate by HPLC, identify by mass spectrometry

• Advantages: Direct detection of pseudouridine, quantitative analysis possible

• Limitations: Requires specialized equipment, doesn't provide position information

• Controls: Compare with unmodified tRNA digests as negative control

Method 2: Site-Specific Detection by Primer Extension

• Procedure: Treat RNA with CMC (N-cyclohexyl-N'-(2-morpholinoethyl)carbodiimide), which specifically modifies pseudouridine and blocks reverse transcriptase

• Advantages: Provides exact position of modification, can be multiplexed

• Limitations: Indirect detection, potential for artifacts

• Controls: Include untreated RNA and known pseudouridine-containing standards

Method 3: Thin-Layer Chromatography (TLC)

• Procedure: "Digest modified tRNA with nuclease P1, and analyze resulting digested products by 2D–TLC. Autoradiographies of the TLC plates revealed the presence of a radiolabeled spot corresponding to pm¹Ψ"

• Advantages: Well-established technique, can distinguish between different modifications

• Limitations: Requires radiolabeling, lower sensitivity than mass spectrometry

• Controls: Include standards for pseudouridine and uridine

Method 4: Nearest Neighbor Analysis

• Procedure: "RNase T2 digestion on radiolabeled tRNA samples confirmed the presence of m¹Ψp only in the tRNA Trp incubated with both Pus10 and TrmY"

• Advantages: Provides sequence context information

• Limitations: Complex procedure, requires specific radiolabeling

• Controls: Include known sequence contexts as standards

Method 5: Absorbance Spectroscopy

• Procedure: Monitor UV spectrum changes (pseudouridine has distinct absorbance properties)

• Advantages: Non-destructive, can be performed in real-time

• Limitations: Less specific than other methods, affected by other modifications

• Controls: Compare with unmodified tRNA and synthetic pseudouridine standards

For comprehensive confirmation, researchers should employ at least two complementary methods, ideally combining a direct detection method (LC-MS/MS) with a position-specific technique (primer extension or nearest neighbor analysis).