Recombinant Rickettsia canadensis tRNA pseudouridine synthase A (truA)

What is tRNA pseudouridine synthase A (truA) and what is its function in Rickettsia canadensis?

tRNA pseudouridine synthase A (truA) is an enzyme (EC 5.4.99.12) that catalyzes the formation of pseudouridine at positions 38-40 in the anticodon stem-loop of tRNAs. In Rickettsia canadensis, this enzyme plays a critical role in post-transcriptional modification of tRNA molecules, which is essential for proper translation fidelity. The enzyme acts by isomerizing specific uridine residues to pseudouridine through a mechanism involving breakage of the N-C glycosidic bond, rotation of the uracil base, and reformation of a C-C glycosidic bond .

The full-length recombinant protein consists of 245 amino acids and has a characteristic sequence that includes several conserved motifs essential for its catalytic activity and substrate recognition .

How does Rickettsia canadensis truA compare structurally and functionally to pseudouridine synthases in other bacterial species?

While specific comparative data for R. canadensis truA is limited in the provided search results, research on Rickettsia evolution provides insights into potential conservation patterns. TruA belongs to a family of pseudouridine synthases that is generally well-conserved across bacterial species, though with variations that may reflect adaptation to different ecological niches.

In comparative genomic studies of Rickettsia species, many housekeeping genes show evidence of recombination between species, while maintaining relatively similar phylogenies . By extension, truA likely follows this pattern of conservation with some species-specific variations. Unlike surface proteins such as rOmpA and rOmpB that show evidence of intense positive selection and rapid diversification between Rickettsia species, intracytoplasmic proteins (like truA would be) typically show lower selective pressures .

What are the optimal storage and handling conditions for Recombinant Rickettsia canadensis truA?

The recombinant Rickettsia canadensis truA should be stored at -20°C for regular storage, and at -20°C or -80°C for extended storage periods. Repeated freezing and thawing cycles should be avoided as they can compromise protein integrity and activity .

For working with the protein:

• Briefly centrifuge the vial before opening to ensure contents are at the bottom

• Reconstitute the protein in deionized sterile water to a concentration of 0.1-1.0 mg/mL

• Add glycerol to a final concentration of 5-50% (with 50% being the standard recommendation)

• Aliquot the solution for long-term storage at -20°C/-80°C

• Working aliquots can be stored at 4°C for up to one week

The shelf life of the liquid form is approximately 6 months at -20°C/-80°C, while the lyophilized form can be stored for about 12 months at the same temperatures .

What expression systems are most effective for producing functional Recombinant Rickettsia canadensis truA?

Based on the product information, mammalian cell expression systems have been successfully used to produce functional Recombinant Rickettsia canadensis truA . This approach likely provides advantages for proper protein folding and potential post-translational modifications.

For researchers developing their own expression protocols, it's worth noting that experiences with other Rickettsia species may provide valuable insights. For instance, successful transformation of Rickettsia typhi has been achieved using electroporation with specific parameters (18-kV/cm field strength, 10 μF capacity, and 600 Ω resistance resulting in a 5.7 ms pulse duration) . While this relates to whole bacterial transformation rather than protein expression, it demonstrates the feasibility of genetic manipulation techniques with Rickettsia species.

When expressing Rickettsia proteins heterologously, researchers should consider:

• Codon optimization for the host system

• Addition of appropriate tags for purification

• Careful selection of expression conditions to enhance solubility

• Verification of enzymatic activity after purification

What are the most reliable methods for assessing the enzymatic activity of Recombinant Rickettsia canadensis truA?

While the search results don't provide specific assays for truA activity, standard methods for pseudouridine synthase activity determination can be applied:

• Tritium Release Assay: This classical approach involves using [5-³H]UTP-labeled tRNA substrates. The enzyme converts the labeled uridine to pseudouridine, releasing tritium into the aqueous phase, which can be measured by scintillation counting.

• HPLC-Based Assays: After incubation with the enzyme, tRNA can be digested to nucleosides and analyzed by HPLC to quantify pseudouridine formation.

• Mass Spectrometry: LC-MS/MS approaches offer high sensitivity for detecting pseudouridine formation in specific tRNA positions.

• Gel-Based Methods: Pseudouridine formation can cause specific mobility shifts in properly designed RNA oligonucleotides under certain electrophoresis conditions.

When establishing an activity assay, researchers should include appropriate controls:

• Heat-inactivated enzyme (negative control)

• Known active pseudouridine synthase (positive control)

• Substrate specificity controls (various tRNA species)

How can researchers effectively measure the substrate specificity of Rickettsia canadensis truA?

To determine the substrate specificity of truA, researchers should consider the following methodological approach:

• Preparation of Various tRNA Substrates:

  • In vitro transcribed tRNAs with different anticodon sequences

  • Naturally purified tRNAs from various organisms

  • Synthetic tRNA fragments focusing on the anticodon stem-loop region

• Comparative Activity Analysis:

  • Parallel enzymatic reactions with different substrates under identical conditions

  • Quantification of pseudouridine formation at specific positions

  • Kinetic analysis (Km, Vmax, kcat) for each substrate

• Mutation Analysis:

  • Test sequence variants of the truA recognition elements

  • Examine the impact of anticodon stem-loop structural variations

• Data Presentation:
  Present results in a comparative table format:

  tRNA Substrate	Specific Activity (nmol/min/mg)	Relative Activity (%)	Km (μM)	kcat (s⁻¹)	kcat/Km (M⁻¹s⁻¹)
  tRNA^Ala	[value]	[value]	[value]	[value]	[value]
  tRNA^Phe	[value]	[value]	[value]	[value]	[value]
  [other tRNAs]	[value]	[value]	[value]	[value]	[value]

How does truA compare to other RNA modification enzymes in the Rickettsia genus?

RNA modification enzymes in bacteria, including those in Rickettsia, play crucial roles in fine-tuning RNA function. Within the Rickettsia genus, analysis of evolutionary patterns can reveal important insights about truA compared to other RNA-modifying enzymes.

Based on studies of gene evolution patterns in Rickettsia, we can infer that truA likely follows patterns similar to other intracytoplasmic proteins. Unlike surface proteins such as rOmpA and rOmpB that show evidence of intense positive natural selection (causing rapid diversification between species), intracytoplasmic proteins like PS120 show low selective constraints but no evidence of positive selection .

A comprehensive phylogenetic analysis of truA sequences across Rickettsia species would help establish:

• The degree of conservation of catalytic and substrate-binding residues

• Whether any regions show evidence of positive selection

• The presence of recombination events in the evolutionary history of the gene

What insights can be gained from comparing the pseudouridylation patterns in Rickettsia canadensis with other bacterial pathogens?

Pseudouridylation patterns in tRNA can vary significantly between bacterial species and may reflect adaptation to different ecological niches and lifestyles. For intracellular pathogens like Rickettsia canadensis, these modifications may be particularly important for adaptation to the host environment.

Comparative analysis should focus on:

• Position-Specific Modifications:

  • Are the same positions modified in tRNAs across different bacterial pathogens?

  • Do obligate intracellular pathogens show similar modification patterns?

• Quantitative Variation:

  • What is the stoichiometry of pseudouridylation at specific positions?

  • How does this vary between growth conditions or infection stages?

• Functional Consequences:

  • How do these modifications affect tRNA stability, codon recognition, and translation accuracy?

  • Are there correlations between modification patterns and pathogen-specific codon usage?

• Evolutionary Implications:

  • Is there evidence for convergent evolution in pseudouridylation patterns among pathogens with similar lifestyles?

  • How do modification patterns correlate with evolutionary relationships?

How can Recombinant Rickettsia canadensis truA be used as a tool for studying bacterial RNA modification systems?

Recombinant Rickettsia canadensis truA provides a valuable tool for investigating RNA modification systems, particularly in hard-to-culture intracellular pathogens. Researchers can utilize this recombinant protein for:

• Comparative Biochemistry:

  • Side-by-side analysis with truA enzymes from other bacterial species

  • Structure-function studies using site-directed mutagenesis

  • Analysis of catalytic mechanisms under different conditions

• Substrate Engineering:

  • Development of modified tRNA substrates to probe enzyme specificity

  • Creation of fluorescent or affinity-tagged tRNA substrates for high-throughput assays

  • Design of inhibitor screening platforms

• Systems Biology Applications:

  • Integration with transcriptomics and proteomics data to understand the global impact of tRNA modifications

  • Analysis of truA-dependent changes in translation efficiency and accuracy

  • Investigation of potential regulatory roles beyond canonical tRNA modification

• Technological Innovations:

  • Development of truA-based RNA labeling techniques

  • Creation of biosensors for studying RNA dynamics in living cells

  • Design of synthetic biology tools leveraging site-specific RNA modification

What is known about the potential role of truA in Rickettsia pathogenesis and host adaptation?

While the search results don't provide direct evidence regarding truA's role in Rickettsia pathogenesis, we can draw informed inferences based on the importance of RNA modifications in bacterial physiology and pathogenesis more broadly.

For intracellular pathogens like Rickettsia canadensis, adaptation to the host environment involves precise regulation of gene expression. RNA modifications, including pseudouridylation, can influence translation efficiency, accuracy, and stress responses—all potentially critical for pathogen survival and virulence.

Several lines of investigation could elucidate truA's role in pathogenesis:

• Comparative Expression Analysis:

  • Examine truA expression levels during different stages of infection

  • Compare expression between virulent and attenuated strains

  • Analyze transcriptional responses to various host environments

• Functional Genomics Approaches:

  • While genetic manipulation of Rickettsia is challenging, techniques developed for related species could be adapted

  • Transformation approaches similar to those used for R. typhi with the pRAM18dRGA plasmid might be applicable

  • Conditional expression systems could help assess the impact of truA activity modulation

• Host Response Analysis:

  • Investigate whether modified tRNAs or the truA protein itself triggers host immune responses

  • Examine potential interactions between truA and host cellular components

• Translation Efficiency Studies:

  • Assess how truA-mediated modifications affect translation of specific Rickettsia virulence factors

  • Analyze codon usage in virulence-associated genes in relation to truA activity

What are common challenges in working with Recombinant Rickettsia canadensis truA and how can they be addressed?

Researchers working with Recombinant Rickettsia canadensis truA may encounter several technical challenges:

• Protein Solubility and Stability Issues:

  • Challenge: Aggregation or precipitation during storage or experimental procedures

  • Solution: Optimize buffer conditions (pH, salt concentration, additives), consider adding stabilizers like glycerol (5-50%) , and avoid repeated freeze-thaw cycles

• Activity Preservation:

  • Challenge: Loss of enzymatic activity during storage or manipulation

  • Solution: Store in small aliquots at -80°C, add reducing agents if necessary, and validate activity periodically with standard assays

• Substrate Availability:

  • Challenge: Obtaining suitable tRNA substrates for activity assays

  • Solution: Consider both in vitro transcribed tRNAs and synthetic oligonucleotides mimicking the anticodon stem-loop

• Specificity Determination:

  • Challenge: Identifying the exact nucleotide positions modified by truA

  • Solution: Employ detailed mapping techniques like primer extension, HPLC-MS/MS, or next-generation sequencing approaches

• Comparison to Native Enzyme:

  • Challenge: Determining if the recombinant protein accurately represents the native enzyme's properties

  • Solution: When possible, compare key parameters with the native enzyme isolated from Rickettsia canadensis

How can researchers integrate truA studies with broader investigations of Rickettsia biology?

Integrating truA studies with broader investigations of Rickettsia biology requires strategic approaches that connect molecular mechanisms to cellular and organismal phenotypes:

• Coordinate with Transformation Studies:

  • Leverage techniques developed for transformation of other Rickettsia species, such as the electroporation protocols used for R. typhi

  • Consider expressing tagged versions of truA to track localization and interactions in vivo

• Connect to Evolutionary Studies:

  • Analyze truA in the context of Rickettsia evolution, considering whether it follows patterns similar to other intracytoplasmic proteins or shows unique evolutionary signatures

  • Determine if recombination events have shaped truA diversity across Rickettsia species

• Link to Host-Pathogen Interaction Studies:

  • Investigate whether truA-mediated tRNA modifications change during different stages of host cell infection

  • Consider how these modifications might influence bacterial adaptation to different host environments

• Develop Integrated Experimental Pipelines:

  • Design workflows that connect in vitro biochemical studies with cellular and infection models

  • Create data integration frameworks to correlate RNA modification patterns with transcriptomic, proteomic, and phenotypic data

• Collaborative Research Approaches:

  • Establish collaborations between structural biologists, biochemists, microbiologists, and computational biologists

  • Develop shared resources and standardized protocols to facilitate comparative studies across different Rickettsia species

What emerging technologies could enhance our understanding of Rickettsia canadensis truA function and regulation?

Several cutting-edge technologies could significantly advance our understanding of truA function and regulation in Rickettsia canadensis:

• CRISPR-Based Approaches:

  • While genetic manipulation of Rickettsia remains challenging, adaptations of CRISPR technologies might enable more precise genetic perturbations

  • CRISPRi approaches could potentially allow for conditional knockdown of truA expression

  • Base editing techniques might permit precise modification of truA sequence without full gene replacement

• Advanced RNA Sequencing Methods:

  • Direct RNA sequencing using nanopore technology can detect modified nucleotides without chemical treatment

  • NAIL-MS (Nucleic Acid Isotope Labeling coupled with Mass Spectrometry) can track dynamic changes in RNA modifications

  • Ribosome profiling could link truA-dependent modifications to translation efficiency changes

• Structural Biology Innovations:

  • Cryo-EM could reveal the structure of truA in complex with its tRNA substrates

  • Hydrogen-deuterium exchange mass spectrometry could map dynamic protein-RNA interactions

  • Integrative structural biology approaches combining multiple techniques could provide comprehensive models of truA function

• Single-Cell and Spatial Technologies:

  • Single-bacterium RNA modification mapping could reveal heterogeneity in truA activity

  • Spatial transcriptomics adapted to host-pathogen systems could localize truA activity during infection

  • Live-cell imaging with RNA modification sensors could track dynamic changes in pseudouridylation

What are promising strategies for developing truA-targeted antimicrobial approaches against Rickettsia infections?

While the search results don't directly address antimicrobial approaches targeting truA, we can outline promising strategies based on understanding of RNA modification enzymes as potential antimicrobial targets:

• Structure-Based Inhibitor Design:

  • Leverage structural information about truA to identify potential binding pockets

  • Design small molecule inhibitors that specifically target Rickettsia truA without affecting human pseudouridine synthases

  • Employ fragment-based drug discovery approaches to identify initial chemical scaffolds

• High-Throughput Screening Platforms:

  • Develop biochemical assays suitable for screening compound libraries

  • Establish cell-based assays that can detect inhibition of truA activity in a cellular context

  • Use phenotypic screening to identify compounds that mimic truA deficiency

• Peptide-Based Inhibitors:

  • Design peptides that interfere with truA-tRNA interactions

  • Develop cell-penetrating peptides that can access the intracellular environment

  • Create peptide mimetics with improved stability and pharmacokinetic properties

• RNA-Based Strategies:

  • Design decoy RNA molecules that compete with natural substrates

  • Develop aptamers that specifically bind and inhibit truA

  • Create modified tRNAs that irreversibly bind to the enzyme

• Validation Approaches:

  • Establish animal models suitable for testing truA-targeted therapeutics

  • Develop reporter systems to monitor truA inhibition in vivo

  • Assess potential for resistance development through in vitro evolution experiments