Recombinant Thermotoga lettingae tRNA pseudouridine synthase A (truA)

What is Thermotoga lettingae tRNA pseudouridine synthase A (truA) and what is its primary function?

Thermotoga lettingae tRNA pseudouridine synthase A (truA) is an enzyme that catalyzes the formation of pseudouridine (Ψ) at specific positions in tRNA molecules. As a member of the tRNA pseudouridine synthase family, truA typically targets positions 38-40 in the anticodon loop of tRNAs. The enzyme functions by isomerizing uridine residues to pseudouridine through C-C bond formation, without breaking the glycosidic bond . This modification enhances tRNA structural stability and translational efficiency, particularly in the thermophilic environment where Thermotoga lettingae thrives at temperatures around 65°C .

What are the optimal storage and handling conditions for recombinant truA?

Based on manufacturer specifications, recombinant Thermotoga lettingae truA should be stored at -20°C for standard storage, or at -80°C for extended storage periods. Working aliquots can be maintained at 4°C for up to one week, but repeated freeze-thaw cycles should be avoided . For reconstitution, the protein should be centrifuged briefly before opening, then reconstituted in deionized sterile water to a concentration of 0.1-1.0 mg/mL with addition of 5-50% glycerol (final concentration) for long-term storage . The shelf life is approximately 6 months for liquid form at -20°C/-80°C and 12 months for lyophilized form at the same temperature range .

What is the structural composition of Thermotoga lettingae truA?

Thermotoga lettingae truA is a full-length protein comprising 245 amino acids with the sequence provided by the manufacturer . The protein contains several conserved domains characteristic of pseudouridine synthases, including catalytic aspartate residues (likely corresponding to positions similar to D48 and D90 in related TruB proteins) and RNA-binding residues (such as K64 in related proteins) that are highly conserved across this family of enzymes . Similar to other pseudouridine synthases like TruB from Thermotoga maritima, which has been crystallized , truA likely contains a catalytic domain responsible for pseudouridylation and an RNA-binding domain that specifically recognizes tRNA substrates.

What are the recommended methods for assessing truA enzymatic activity in vitro?

For assessing Thermotoga lettingae truA enzymatic activity, researchers should employ the following methodological approaches:

a) CMC-primer extension assay: This involves treatment of RNA with N-cyclohexyl-N'-(2-morpholinoethyl)carbodiimide metho-p-toluenesulfonate (CMC), which specifically binds to pseudouridine residues. Subsequent alkaline treatment removes CMC from all nucleotides except pseudouridine. Primer extension using reverse transcriptase then stops at the CMC-modified pseudouridine sites, creating specific termination products that can be visualized by gel electrophoresis .

b) In vitro enzymatic assays: Using purified recombinant truA and appropriate tRNA substrates (typically tRNA lacking pseudouridine modifications), reactions should be conducted at 55-65°C (optimal temperature for T. lettingae enzymes) in a buffer system maintaining pH 7.0, which aligns with the optimal pH for growth of T. lettingae .

c) Substrate specificity determination: Activity should be tested against various tRNA isoacceptors to assess substrate range, as well as with RNA point mutations at target sites to identify critical recognition elements.

For all assays, appropriate controls must be included, particularly catalytically inactive variants (D48A, D90A equivalents) that can distinguish enzyme-dependent activity from background .

How can researchers effectively express and purify recombinant Thermotoga lettingae truA?

The recommended protocol for expression and purification of recombinant Thermotoga lettingae truA involves:

a) Expression system: Overexpression in E. coli strains lacking endogenous CCA-adding enzyme (e.g., JM109(DE3) cca:cam or E. coli CA244 cca:cam) using a vector containing an N-terminal His-tag for purification purposes .

b) Culture conditions: Grow transformed E. coli in LB medium at 37°C with appropriate antibiotics until OD600 reaches 0.6, then induce with IPTG (1 mM final concentration) for 3-5 hours at 37°C or overnight at room temperature to optimize protein folding .

c) Purification steps:

• Cell lysis and initial purification according to established protocols for thermostable His-tagged proteins

• Affinity chromatography using Ni-NTA resin

• Further purification to apparent homogeneity (typically 7-fold purification is reported for similar thermostable enzymes)

• Quality assessment by SDS-PAGE (target >85% purity)

This approach yields functional recombinant truA suitable for structural and biochemical studies, with proper folding confirmed by enzymatic activity assays.

How should researchers design mutation studies to investigate truA function?

When designing mutation studies for Thermotoga lettingae truA, researchers should target specific residues based on conservation and known functional roles in related enzymes:

a) Catalytic residues: Create variants with mutations in the predicted catalytic aspartate residues (equivalents to D48 and D90 identified in related TruB proteins) . These mutations should abolish enzymatic activity while potentially maintaining RNA binding.

b) RNA binding residues: Target conserved lysine residues (equivalent to K64 in related enzymes) that are critical for RNA substrate recognition. These mutations should disrupt RNA binding while leaving the catalytic center intact.

c) Experimental validation approach:

d) Comparative analysis: Compare the effects of equivalent mutations across pseudouridine synthases from mesophilic and thermophilic organisms to identify thermoadaptation mechanisms .

This systematic mutation approach can separate RNA binding from catalytic functions and identify key residues responsible for thermostability and substrate specificity.

How can researchers investigate potential non-canonical functions of Thermotoga lettingae truA?

Investigating non-canonical functions of Thermotoga lettingae truA requires approaches that extend beyond traditional enzymatic characterization:

a) Separation of enzymatic and binding activities:

• Generate catalytically inactive mutants (targeting conserved D48 and D90 equivalent residues) that retain RNA binding capability

• Create binding-deficient mutants (targeting K64 equivalent residue) to distinguish between catalytic and structural roles

• Compare effects of these variants in functional assays

b) RNA interaction profiling:

• Perform RNA immunoprecipitation followed by sequencing (RIP-seq) to identify all RNA binding partners

• Use CLIP-seq methods to map direct RNA-protein interaction sites with nucleotide resolution

• Compare with findings from studies of related pseudouridine synthases like TruB1, which has been shown to regulate miRNA maturation independently of its enzymatic activity

c) Non-tRNA substrate validation:

• Test activity on predicted non-canonical RNA substrates using in vitro pseudouridylation assays

• Map pseudouridine sites using CMC-based methods

• Perform functional analysis of pseudouridylated vs. non-pseudouridylated RNA variants

This approach has identified unexpected functions in related enzymes - for example, TruB1 was found to selectively enhance the interaction between pri-let-7 and the microprocessor DGCR-8, promoting miRNA maturation independently of its pseudouridylation activity .

How does the thermal stability of Thermotoga lettingae truA compare to other pseudouridine synthases, and what are the structural determinants?

Thermotoga lettingae truA, originating from an organism with an optimal growth temperature of 65°C , exhibits significant thermostability compared to mesophilic counterparts. The structural basis for this thermostability can be investigated through:

a) Comparative analysis with other Thermotoga enzymes:

b) Structural determinants of thermostability:

• Increased number of salt bridges and hydrophobic interactions

• More compact protein folding with shorter loop regions

• Higher proportion of charged amino acids on the protein surface

c) Experimental approaches:

• Differential scanning calorimetry to determine melting temperatures

• Activity assays at various temperatures to establish the temperature optimum

• Circular dichroism spectroscopy to monitor temperature-dependent structural changes

• Targeted mutagenesis of residues predicted to contribute to thermostability

The insights from these studies can reveal the molecular adaptations that enable truA to function at elevated temperatures and inform protein engineering strategies for enhancing thermostability of other enzymes.

What approaches can be used to study the role of truA in stress response and adaptation in Thermotoga lettingae?

Investigating the role of truA in stress response and adaptation in Thermotoga lettingae requires specialized approaches:

a) Stress response characterization:

• Profile tRNA modifications under various stress conditions (heat shock, cold shock, nutrient limitation)

• Correlate pseudouridylation levels with translation efficiency during stress

• Compare responses to related Thermotoga species that differ in optimal growth temperatures (T. maritima: 80°C, T. lettingae: 65°C)

b) Comparative genomics:

• Analyze truA conservation across Thermotoga species with different optimal growth temperatures

• Examine genomic context and potential co-regulated genes

• Compare with other thermophiles to identify convergent adaptations

c) Experimental design for stress studies:

Unlike other Thermotoga species that primarily grow at 75-80°C, T. lettingae has a lower optimal temperature (65°C) , making it an excellent model for studying temperature adaptation within this genus. This comparative approach can reveal how truA activity contributes to thermoadaptation and stress response in these extremophiles.

How should researchers interpret discrepancies in truA activity data between in vitro and in vivo experiments?

When facing discrepancies between in vitro and in vivo truA activity data, researchers should consider several factors specific to thermophilic enzymes:

a) Temperature considerations:

• In vitro assays may be conducted at different temperatures than the organism's optimal growth temperature (65°C for T. lettingae)

• The enzyme's activity profile may exhibit a narrower temperature range than expected

• Similar enzymes like TLDex from T. lettingae show maximum activity at specific temperatures (55-60°C) with rapid decline outside this range

b) Methodological approach to reconciliation:

• Validate enzyme activity across a temperature gradient (25-80°C)

• Consider pH effects, as T. lettingae has an optimal pH of 7.0

• Examine salt requirements, as T. lettingae grows optimally with 1.0% NaCl

• Test whether metal ions affect activity (as observed with related enzymes)

c) Specific considerations for thermophilic enzymes:

• Thermostable enzymes often exhibit unusual kinetic features, such as negative cooperative behavior observed with other T. lettingae enzymes

• Protein folding and substrate recognition may be temperature-dependent

• The cellular environment of thermophiles includes stabilizing solutes and unique ion concentrations

By systematically addressing these factors, researchers can develop more accurate models of truA function that bridge in vitro biochemical data with in vivo biological relevance.

What controls should be included when studying truA specificity and activity?

When studying Thermotoga lettingae truA specificity and activity, the following controls should be included:

a) Negative controls:

• No-enzyme control to assess background modifications

• Heat-inactivated enzyme (typically 95°C for 10 minutes) to confirm enzyme-dependent activity

• Mutated truA variants (D48A and D90A equivalents) with disrupted catalytic activity

• Non-substrate RNAs lacking the target modification sites

b) Positive controls:

• Known tRNA substrates with well-characterized pseudouridylation sites

• Pre-modified pseudouridine-containing RNA to establish detection limits

c) Specificity controls:

• Various tRNA isoacceptors to assess substrate range

• tRNA with point mutations at target sites

• Chimeric RNAs to identify minimal recognition elements

d) Technical controls:

• Temperature gradient experiments (particularly important for thermophilic enzymes)

• pH variations to establish optimal conditions, considering T. lettingae's optimal pH of 7.0

• Metal ion effects, as some divalent cations can significantly impact enzyme activity (as seen with Fe²⁺ and Ag²⁺ inhibition of related T. lettingae enzymes)

These controls ensure that observed pseudouridylation is specifically attributed to truA activity and help characterize the enzyme's substrate requirements and catalytic properties in the context of its thermophilic nature.

How can researchers overcome challenges in structural characterization of Thermotoga lettingae truA?

Structural characterization of Thermotoga lettingae truA presents several challenges that require specific methodological approaches:

a) Protein crystallization strategies:

• Screen crystallization conditions at various temperatures, particularly near T. lettingae's optimal growth temperature (65°C)

• Use surface entropy reduction through site-directed mutagenesis

• Consider the successful approach used for T. maritima TruB crystallization: hanging-drop vapor-diffusion method in specific buffer conditions (100 mM citrate pH 3.5, 200 mM Li₂SO₄, 20% glycerol, 13% PEG 8000)

• Co-crystallize with substrate tRNA or substrate analogs to stabilize conformation

b) Alternative structural approaches:

c) RNA-protein complex characterization:

• Use tRNA binding assays to establish optimal conditions for complex formation

• Apply molecular dynamics simulations at elevated temperatures

• Consider the workflow used for related thermostable RNA-modifying enzymes like T. maritima TruB, which successfully yielded diffraction-quality crystals with resolution to 2.0 Å

By combining these approaches and leveraging the experience from structural studies of related thermophilic RNA-modifying enzymes, researchers can overcome the inherent challenges in structural characterization of Thermotoga lettingae truA.

How might Thermotoga lettingae truA be utilized in synthetic biology applications?

Thermotoga lettingae truA offers several valuable properties for synthetic biology applications, particularly related to its thermostability:

a) RNA engineering applications:

• Site-specific introduction of pseudouridine to enhance RNA stability at elevated temperatures

• Development of thermostable RNA devices for high-temperature bioprocesses

• Creation of modified tRNAs for specialized translation in thermophilic expression systems

b) Enzyme engineering opportunities:

• Creation of chimeric pseudouridine synthases with altered specificity, similar to the approach used with A-adding enzymes from Thermotoga species

• Development of truA variants with expanded substrate range, leveraging the natural ability of related pseudouridine synthases to recognize non-canonical substrates

• Engineering bifunctional enzymes by combining domains from different Thermotoga RNA-modifying enzymes

c) Thermostable tool development:

• Incorporation into high-temperature in vitro transcription-translation systems

• Development of heat-resistant RNA-processing modules

• Creation of thermophilic cell-free systems with enhanced stability

These applications leverage truA's thermostability (functioning at 65°C) and specific RNA modification capability to address challenges in synthetic biology applications requiring robust performance at elevated temperatures.

What are the key areas for future research on Thermotoga lettingae truA?

Future research on Thermotoga lettingae truA should focus on several critical areas:

a) Comprehensive structural characterization:

• Determine high-resolution structure through X-ray crystallography or cryo-EM

• Elucidate the structural basis for thermostability by comparison with mesophilic homologs

• Characterize conformational changes during catalysis

• Compare with the available structural data from related thermophilic pseudouridine synthases

b) Complete substrate profiling:

• Identify the full set of RNA targets in vivo

• Map all pseudouridylation sites in the T. lettingae transcriptome

• Determine the minimal RNA elements required for recognition

• Compare substrate specificity with other Thermotoga species that grow at different optimal temperatures

c) Evolutionary analysis:

• Investigate the evolutionary relationship between truA proteins across the Thermotoga genus, which spans a temperature optimum range from 65°C to 80°C

• Examine potential horizontal gene transfer events, particularly important in Thermotogales

• Analyze co-evolution of tRNA substrates and modification enzymes in thermophiles

d) Functional characterization beyond canonical activity:

• Investigate potential non-canonical functions similar to those identified for TruB1, which promotes miRNA processing independently of its enzymatic activity

• Explore the role of truA in stress response and adaptation in thermophilic environments

• Study the impact of tRNA modifications on translation efficiency at high temperatures

These research directions will significantly advance our understanding of RNA modification biology in thermophilic organisms and potentially reveal novel principles of thermoadaptation at the molecular level.

How can researchers investigate the evolutionary relationship between Thermotoga lettingae truA and other pseudouridine synthases?

Investigating the evolutionary relationships between Thermotoga lettingae truA and other pseudouridine synthases requires an integrated approach:

a) Phylogenetic analysis:

• Construct comprehensive phylogenetic trees including representatives across all pseudouridine synthase families

• Focus on the unique evolutionary position of Thermotoga species, which differ in optimal growth temperatures (T. lettingae: 65°C vs. other species: 75-80°C)

• Analyze the relationship between temperature adaptation and sequence evolution

b) Structural comparison:

• Compare truA with the characterized structures of related enzymes like T. maritima TruB

• Identify structural adaptations specific to thermophilic environments

• Examine conservation of catalytic residues and substrate binding motifs

c) Functional evolution:

• Compare substrate specificity across homologs from different Thermotoga species

• Test activity under various conditions (temperature, pH, salt) to identify adaptive shifts

• Analyze potential conserved non-canonical functions like those observed in TruB1

d) Comparative genomics within Thermotoga:

• Analyze genomic context of truA genes across Thermotoga species with different optimal growth temperatures

• Examine the core genome of hyperthermophilic Thermotoga species (which comprises about 75% of their genomes)

• Identify co-evolved genes that may function with truA in RNA modification pathways

This evolutionary perspective can provide insights into how pseudouridine synthases have adapted to function at different temperature optima within the Thermotoga genus, potentially revealing principles of enzyme thermoadaptation that can be applied to protein engineering efforts.