Recombinant Dinoroseobacter shibae tRNA pseudouridine synthase A (truA)

What is the biological function of tRNA pseudouridine synthase A in Dinoroseobacter shibae?

tRNA pseudouridine synthase A (truA) in D. shibae, like its homologs in other bacterial species, catalyzes the isomerization of uridine to pseudouridine at positions 38, 39, and/or 40 in the anticodon stem-loop of tRNA. This enzyme belongs to the pseudouridine synthase family, which shares a conserved catalytic mechanism featuring an essential aspartate residue in the active site .

Pseudouridylation in the anticodon stem-loop enhances translational accuracy by:

• Stabilizing the ASL structure through improved base stacking

• Influencing codon-anticodon interactions

• Contributing to proper tRNA folding and function

In the marine α-proteobacterium D. shibae, which is known for its ecological significance in marine environments and its complex gene regulatory networks, RNA modifications likely play important roles in adaptation to environmental conditions .

How does the structure of D. shibae truA compare with other characterized bacterial tRNA pseudouridine synthases?

While the specific structure of D. shibae truA has not been fully characterized in the provided research, we can make informed comparisons based on structural studies of homologous enzymes:

The crystal structure of TruA from Thermus thermophilus revealed "remarkably flexible structural features in the tRNA-binding cleft," which are likely responsible for primary tRNA interaction . Similar to other pseudouridine synthases, the D. shibae truA likely contains a conserved active site aspartate that is essential for catalysis .

What experimental approaches should be used to characterize the substrate specificity of D. shibae truA?

To characterize the substrate specificity of D. shibae truA, researchers should implement a multi-faceted approach:

Biochemical Characterization:

• In vitro pseudouridylation assays: Using radiolabeled or fluorescently labeled tRNA substrates to monitor modification efficiency

• Site-directed mutagenesis: Altering the target uridines to determine specificity at positions 38, 39, and 40

• Competition assays: Using various tRNA substrates to determine relative affinities

Structural Analysis:

• Crystallography: Co-crystallization with tRNA substrates, similar to the approaches used with TruB that revealed "a combination of rigid docking and induced fit" mechanism

• Cryo-EM: For visualizing enzyme-substrate complexes

• NMR studies: To identify dynamic interactions during catalysis

Computational Approaches:

• Homology modeling: Based on known structures of TruA from other organisms

• Molecular docking simulations: To predict substrate binding modes

• Sequence alignment analysis: To identify conserved residues important for specificity

This comprehensive approach would reveal how D. shibae truA achieves its substrate "promiscuity" - the ability to modify uridines at three different positions in the ASL of various tRNAs .

How should researchers optimize expression and purification of recombinant D. shibae truA?

Based on the available product information and general recombinant protein methodologies, the following protocol is recommended:

Expression Systems:

• E. coli: BL21(DE3) or Rosetta strains are suitable for expression

• Yeast: Alternative expression system if E. coli produces inclusion bodies

Expression Conditions:

• Temperature: 16-18°C after induction to enhance solubility

• Induction: 0.1-0.5 mM IPTG, depending on expression system

• Duration: 16-20 hours for optimal yield

Purification Strategy:

• Cell lysis: Sonication or French press in buffer containing 50 mM Tris-HCl pH 8.0, 300 mM NaCl, 10% glycerol

• Initial purification: Affinity chromatography using appropriate tag (His-tag commonly used)

• Secondary purification: Size exclusion chromatography

• Quality control: SDS-PAGE analysis with expected purity >85%

What is the catalytic mechanism of truA and how can it be experimentally validated?

The catalytic mechanism of pseudouridine synthases involves several key steps:

• Substrate binding: The enzyme binds tRNA, inducing conformational changes in both the enzyme and tRNA

• Base flipping: The target uridine is flipped out of the RNA helix into the active site

• C-C bond cleavage: The glycosidic bond between uracil and ribose is broken

• Base rotation: The uracil is rotated 180°

• C-C bond formation: A new C-C bond forms between C5 of uracil and C1' of ribose

• Product release: The enzyme releases the modified tRNA

The conserved aspartate residue in the active site is crucial for this mechanism .

Experimental Validation Approaches:

• Site-directed mutagenesis: Mutating the conserved aspartate to assess its role

• Mechanism-based inhibitors: Using C5-substituted uridine analogs that trap reaction intermediates

• Isotope labeling: Tracking the movement of specific atoms during catalysis

• Time-resolved crystallography: Capturing enzyme-substrate complexes at different reaction stages

• Quantum mechanics/molecular mechanics (QM/MM) simulations: Computational modeling of the reaction pathway

Research on other truA enzymes has revealed that the enzyme undergoes "significant conformational changes on binding to its substrate," including "the ordering of the 'thumb loop'" and a "10° hinge movement of the C-terminal domain" .

How does tRNA flexibility affect truA substrate recognition and catalysis?

The flexibility of tRNA, particularly in the anticodon stem-loop (ASL), is critical for truA function. Based on structural studies of truA-tRNA complexes:

Key Findings on tRNA Flexibility and truA Function:

• Site promiscuity mechanism: TruA "utilizes the intrinsic flexibility of the ASL for site promiscuity"

• Substrate selection: TruA appears to "select against intrinsically stable tRNAs to avoid their overstabilization through pseudouridylation"

• Conformational changes: The tRNA structure must undergo conformational changes to access the deeply buried active site aspartate residue

• Balance requirement: TruA maintains "the balance between the flexibility and stability required for its biological function"

Experimental Approaches to Study this Relationship:

• SHAPE analysis: To measure tRNA flexibility before and after modification

• Single-molecule FRET: To monitor dynamic conformational changes during enzyme-substrate interactions

• Comparative analysis: Using tRNAs with varying degrees of intrinsic stability as substrates

• Molecular dynamics simulations: To model the conformational landscape of different tRNAs

Understanding this relationship is crucial as it reveals how truA achieves its remarkable ability to modify multiple positions (38, 39, and/or 40) across various tRNAs despite their sequence diversity .

What true experimental design approaches should be used to study D. shibae truA function in vivo?

To study D. shibae truA function in vivo, researchers should implement true experimental designs that control for extraneous variables through randomization and incorporate control groups .

Experimental Design Framework:

• Gene Knockout/Complementation Studies:

  • Generate a truA deletion mutant in D. shibae

  • Create complementation strains with wild-type and mutant versions

  • Include proper controls (wild-type strain, vector-only control)

  • Measure pseudouridylation levels using mass spectrometry or primer extension

• Phenotypic Analysis:

  • Growth rate measurements under various conditions

  • Antibiotic sensitivity assays (translational fidelity)

  • Stress response studies (temperature, pH, salinity)

  • Competition experiments with wild-type strain

• Translational Fidelity Assessment:

  • Reporter gene systems to measure miscoding events

  • Ribosome profiling to assess global translation

  • Pulse-labeling experiments to measure protein synthesis rates

• RNA Structure and Stability Analysis:

  • In vivo SHAPE-Seq to assess tRNA structural changes

  • Northern blotting to measure tRNA half-lives

  • tRNA aminoacylation levels

Each experiment should follow the principles of true experimental design, including random assignment to treatment groups, appropriate controls, and statistical analysis of results to determine significance .

How can researchers distinguish between the functions of different tRNA modification enzymes in D. shibae?

Distinguishing between the functions of different tRNA modification enzymes in D. shibae requires systematic approaches:

Methodological Framework:

• Combinatorial Gene Deletions:

  • Create single and multiple deletion mutants of various tRNA modification enzymes

  • Perform complementation studies with enzymes from other species

  • Analyze epistatic relationships between modifications

• Modification Mapping:

  • Use liquid chromatography-mass spectrometry (LC-MS/MS) to catalog all tRNA modifications

  • Apply high-throughput sequencing methods (e.g., Ψ-seq) for transcriptome-wide mapping

  • Perform comparative analysis between wild-type and mutant strains

• tRNA-Specific Analysis:

  • Develop primer extension assays specific for D. shibae tRNAs

  • Use gel mobility shift assays to detect structural changes in modified vs. unmodified tRNAs

  • Create tRNA overexpression systems to test modification saturation effects

• Biochemical Competition Assays:

  • Test whether different modification enzymes compete for the same tRNA substrates

  • Establish order of modifications using in vitro sequential modification assays

  • Measure enzyme kinetics with pre-modified substrates

This systematic approach allows researchers to establish the unique and overlapping functions of different tRNA modification enzymes in D. shibae, contributing to our understanding of the complex RNA modification landscape in this organism.

What evolutionary insights can be gained from studying truA in Roseobacter group bacteria?

Studying truA in the Roseobacter group, including D. shibae, provides valuable evolutionary insights:

Evolutionary Significance:

• Conservation across domains: The pseudouridine synthase family is highly conserved across bacteria, archaea, and eukaryotes, suggesting fundamental importance

• Horizontal gene transfer: D. shibae demonstrates efficient conjugation and gene transfer capabilities that may influence modification enzyme evolution

• Adaptive significance: RNA modifications may contribute to the ecological success of Roseobacter group bacteria in marine environments

Research Approaches:

• Comparative Genomics:

  • Analyze truA sequences across the Roseobacter group

  • Identify conserved residues vs. lineage-specific adaptations

  • Map genomic context of truA genes to detect co-evolution patterns

• Phylogenetic Analysis:

  • Construct phylogenetic trees of truA sequences

  • Compare with species phylogeny to detect horizontal gene transfer events

  • Use tools like Gubbins to identify recombination events in truA evolution

• Functional Comparison:

  • Test substrate specificity of truA enzymes from different Roseobacter species

  • Analyze modification patterns across the clade

  • Perform complementation studies across species boundaries

• Ecological Correlation:

  • Correlate truA sequence variation with ecological niches

  • Investigate environmental conditions that may select for specific truA variants

  • Test temperature adaptations of truA enzymes from bacteria in different thermal niches

The Roseobacter group represents an excellent model for studying enzyme evolution due to their "conspicuous wealth of extrachromosomal replicons" and evidence of horizontal gene transfer as "a major evolutionary driving force" .

How can researchers utilize structural information to engineer D. shibae truA with novel specificities?

Engineering D. shibae truA with novel specificities requires detailed structural understanding and rational design approaches:

Strategic Framework for Engineering:

• Structure Determination Prerequisites:

  • Solve the crystal structure of D. shibae truA alone and in complex with RNA

  • Identify key residues involved in substrate recognition

  • Map the active site architecture in detail

• Computational Design Approaches:

  • Perform molecular docking with various RNA substrates

  • Use molecular dynamics simulations to predict dynamic interactions

  • Apply computational alanine scanning to identify critical binding residues

• Rational Engineering Strategies:

  • Target the RNA-binding cleft that contains "charged residues occupying the intermediate positions"

  • Modify the "thumb loop" that interacts with the RNA hairpin loop

  • Engineer the active site periphery while preserving the conserved catalytic aspartate

• Directed Evolution Methods:

  • Develop high-throughput screening assays for pseudouridylation activity

  • Create truA variant libraries through random mutagenesis

  • Perform rounds of selection for desired specificities

• Validation Techniques:

  • In vitro activity assays with various RNA substrates

  • Structural characterization of engineered variants

  • In vivo functional complementation tests

This engineering approach could potentially create truA variants with applications in synthetic biology, RNA labeling, or as tools for studying RNA modification pathways.

What is the relationship between truA function and horizontal gene transfer in D. shibae?

D. shibae shows remarkable capabilities for horizontal gene transfer, and RNA modifications may play important roles in this process:

Potential Relationships:

• Plasmid Transfer and RNA Modifications:

  • D. shibae has been shown to transfer its 191-kb and 126-kb plasmids between different Roseobacter species

  • tRNA modifications may influence the expression of genes involved in conjugation

  • The type IV secretion systems (T4SS) encoded on these plasmids are regulated by quorum sensing systems

• Translational Fidelity During Gene Transfer:

  • RNA modifications ensure accurate translation of newly acquired genes

  • truA function may be particularly important for expression of foreign genetic material

  • Pseudouridylation could stabilize tRNAs under stress conditions associated with conjugation

• Regulatory Connections:

  • The expression of T4SS in D. shibae is regulated by quorum sensing signals

  • RNA modifications might influence regulatory RNA function in these pathways

  • Global translation effects of truA could impact the expression of conjugation machinery

• Experimental Approaches:

  • Measure conjugation frequencies in truA mutants vs. wild-type strains

  • Analyze tRNA modification patterns during conjugation events

  • Test whether truA mutations alter the expression of genes involved in horizontal gene transfer

Understanding this relationship could provide insights into how RNA modifications contribute to bacterial genome plasticity and evolution, particularly in the Roseobacter group where horizontal gene transfer appears to be "an important mechanism" that may "contribute to its ecological success" .

How do recombination events in D. shibae impact the evolution of truA and other RNA modification enzymes?

Recombination events can significantly influence the evolution of RNA modification enzymes like truA in D. shibae:

Recombination Effects on truA Evolution:

• Homologous Recombination:

  • Recombination could introduce sequence diversity in truA

  • D. shibae and other Roseobacter species show evidence of "efficient exchange of ECRs even between phylogenetically only distantly related mating partners"

  • Tools like Gubbins can identify "loci containing elevated densities of base substitutions suggestive of horizontal sequence transfer"

• Gene Conversion Events:

  • May homogenize truA sequences within bacterial populations

  • Could spread beneficial mutations across strains

  • Potentially maintain conserved catalytic domains while allowing variation in substrate recognition regions

• Recombination Hotspots:

  • Regions near the terminus of replication (ter) in D. shibae show enrichment in DNA transferred via outer membrane vesicles

  • Position of truA relative to such hotspots may influence its evolutionary trajectory

  • Site-specific recombinases like XerCD may influence these patterns

• Research Approaches:

  • Perform population genomics studies of truA sequences across D. shibae isolates

  • Use Gubbins or similar tools to detect recombination events in the truA locus

  • Compare synonymous vs. non-synonymous substitution rates to detect selection pressure

  • Analyze the genomic context of truA for evidence of recent recombination events

Understanding these dynamics provides insight into how essential RNA modification enzymes evolve while maintaining their critical functions in cellular physiology.