Recombinant Agrobacterium vitis tRNA pseudouridine synthase A (truA)

What is the fundamental function of tRNA pseudouridine synthase A (truA) in Agrobacterium vitis?

tRNA pseudouridine synthase A (truA) in Agrobacterium vitis, similar to its homologs in other bacterial species, catalyzes the conversion of specific uridine residues to pseudouridine (Ψ) in the anticodon stem loop (ASL) of tRNAs. Specifically, truA targets uridines at positions 38, 39, and/or 40 of tRNAs with highly divergent sequences and structures. This modification is critical for translational accuracy and efficiency, as it alters the structural properties of the tRNA molecules to optimize their function in protein synthesis .

The enzyme operates through a mechanism that differs from other RNA-modifying enzymes, utilizing the intrinsic flexibility of the ASL for site recognition. This modification strategy helps maintain the delicate balance between flexibility and stability required for tRNA biological function .

How does A. vitis truA differ structurally and functionally from truA in E. coli and other bacteria?

While specific structural comparisons between A. vitis truA and E. coli truA are not directly addressed in the available data, research on E. coli truA provides insights into probable similarities and differences. The E. coli enzyme modifies multiple tRNAs (approximately 17 different tRNAs) with divergent sequences in the ASL region .

A notable feature of truA is its ability to modify nucleotides that are spatially distant (up to 15 Å apart) using a single active site. The enzyme's active site is predominantly hydrophobic and relatively large, which likely contributes to its base recognition mechanism that appears to be independent of specific nucleotide identity .

Comparative analysis would require experimental determination of the A. vitis truA crystal structure and detailed biochemical studies to identify species-specific characteristics that might relate to A. vitis biology and its interaction with plant hosts.

What expression systems are most effective for producing recombinant A. vitis truA?

While the search results don't specifically address expression systems for A. vitis truA, researchers typically employ bacterial expression systems for recombinant production of pseudouridine synthases. The methodological approach would include:

• Vector selection: pET-based expression vectors in E. coli BL21(DE3) or similar strains are commonly used for recombinant expression of bacterial enzymes.

• Optimization parameters:

  • Induction conditions (IPTG concentration, temperature, and duration)

  • Growth media composition

  • Codon optimization for E. coli if expression yields are low

  • Solubility enhancement through fusion tags (His-tag, MBP, GST)

• Purification strategy:

  • Initial capture using affinity chromatography

  • Secondary purification via ion exchange or size exclusion chromatography

  • Optional tag removal depending on downstream applications

Researchers should evaluate multiple expression conditions to determine optimal parameters for obtaining enzymatically active A. vitis truA.

What crystallization techniques are most successful for structural analysis of A. vitis truA-tRNA complexes?

Based on successful approaches with E. coli truA-tRNA complexes, researchers should consider:

• Sample preparation:

  • High-purity recombinant truA (>95% by SDS-PAGE)

  • In vitro transcribed or native tRNA substrates

  • Complex formation by incubation at physiologically relevant conditions

• Crystallization screening:

  • Initial broad screening using commercial sparse matrix kits

  • Optimization of promising conditions with varying:

    • Protein:tRNA ratios (typically 1:1.1 to 1:1.5 molar ratio)

    • Precipitant concentration

    • pH values

    • Additives that promote crystal formation

• Crystal handling considerations:

  • Cryoprotection conditions that preserve diffraction quality

  • Data collection strategies for RNA-protein complexes

Previous studies with E. coli truA yielded crystals in multiple forms that captured different conformational states of the enzyme-substrate complex . Similar approaches may be applicable to A. vitis truA, while accounting for potential differences in protein behavior during crystallization.

How can site-directed mutagenesis be employed to investigate the catalytic mechanism of A. vitis truA?

Site-directed mutagenesis is a powerful approach to investigate truA's catalytic mechanism:

• Target residue selection:

  • Catalytic aspartate residue (equivalent to Asp60 in E. coli truA)

  • Residues involved in tRNA binding and base flipping

  • Residues that may participate in A. vitis-specific functions

• Experimental design:

  • Create alanine substitutions for putative catalytic residues

  • Generate conservative substitutions to probe specific interactions

  • Express mutant proteins under identical conditions as wild-type

• Functional assessment:

  • In vitro pseudouridylation assays using radiolabeled or fluorescently labeled tRNA substrates

  • Binding affinity measurements using isothermal titration calorimetry or surface plasmon resonance

  • Structural analysis of mutant-tRNA complexes

As observed with the D60A mutant in E. coli truA, certain mutations can increase tRNA binding affinity while abolishing catalytic activity, providing valuable mechanistic insights .

What biochemical assays can quantitatively measure A. vitis truA activity and substrate specificity?

Several complementary assays can be employed to characterize A. vitis truA activity:

• Tritium release assay:

  • Principle: Monitoring release of tritium from [5-³H]-UTP-labeled tRNA during pseudouridine formation

  • Advantages: Quantitative, established method

  • Limitations: Requires radioactive materials, indirect measure

• HPLC-based nucleoside analysis:

  • Principle: Enzymatic digestion of modified tRNA followed by HPLC separation and quantification of pseudouridine

  • Advantages: Direct measurement, no radioisotopes required

  • Protocol: Digest tRNA with nuclease P1 and alkaline phosphatase, separate nucleosides by reverse-phase HPLC

• Mass spectrometry approaches:

  • Principle: Detection of mass shift in oligonucleotides containing pseudouridine

  • Advantages: High sensitivity, can map modification sites precisely

  • Applications: LC-MS/MS for exact position mapping

• Substrate competition assays:

  • Purpose: Determine relative substrate preferences

  • Design: Competition between different tRNA species for limited enzyme

These methods collectively provide comprehensive characterization of enzyme kinetics, substrate specificity, and modification patterns.

How does the ASL flexibility influence target site selection by A. vitis truA?

The intrinsic flexibility of the anticodon stem loop (ASL) appears central to truA's site selection mechanism:

• Structural basis:

  • TruA utilizes the natural flexibility of the ASL to access target uridines

  • Crystal structures of E. coli truA-tRNA complexes reveal that the enzyme captures different conformational states of the ASL

  • Three distinct conformations observed: initial docking, intermediate bending toward active site, and target base flipping

• Mechanistic implications:

  • The enzyme appears to select against intrinsically stable tRNAs to avoid their overstabilization through pseudouridylation

  • This selection mechanism maintains the balance between flexibility and stability required for tRNA function

  • Base flipping occurs regardless of nucleotide identity at target positions

• Experimental approach to study in A. vitis truA:

  • Comparative analysis of substrate tRNAs with varying ASL stability

  • Molecular dynamics simulations of A. vitis truA-tRNA complexes

  • NMR studies to capture dynamic aspects of the interaction

This mechanism differs significantly from other pseudouridine synthases like TruB, which recognizes conserved sequences rather than exploiting structural flexibility .

What is the relationship between A. vitis truA and virulence or host specificity in grapevine infections?

While the search results don't directly address the relationship between truA and A. vitis virulence, we can propose research approaches to investigate this connection:

• Potential roles in virulence:

  • Translation optimization under host stress conditions

  • Regulation of virulence factor expression through translational control

  • Adaptation to host environment during infection

• Experimental approaches:

  • Construction of truA knockout mutants in A. vitis

  • Virulence assessment using grape wound inoculation models

  • Transcriptome and proteome analysis of wild-type versus truA mutants during infection

  • Complementation studies to confirm phenotype attribution

• Host specificity considerations:

  • Comparison with non-tumorigenic strains like F2/5 that show host-specific biological control

  • Analysis of truA sequence and activity across A. vitis strains with different host ranges

  • Investigation of potential interaction with host defense mechanisms

This research direction would be particularly valuable as A. vitis is known to cause crown gall disease specifically in grapevines, and strain F2/5 demonstrates host-specific biological control activity .

How do the substrate recognition mechanisms of A. vitis truA compare with those of E. coli and other bacterial truA enzymes?

Based on E. coli truA research findings, we can anticipate certain features of A. vitis truA and design comparative studies:

The study of E. coli truA revealed its unusual ability to modify multiple positions using a single active site and its capacity to accommodate different nucleotides at target positions. Determining whether A. vitis truA shares these properties would provide insights into the evolution and specialization of this enzyme family .

What role might truA play in the ecological adaptation of A. vitis to grapevine compared to related Agrobacterium species?

This question addresses the potential specialized function of truA in A. vitis:

• Ecological considerations:

  • A. vitis survives systemically in grapevines, unlike many other Agrobacterium species

  • Adaptation to grapevine-specific environmental conditions may involve specialized tRNA modifications

  • Potential role in regulating expression of host-specific virulence factors

• Comparative analysis approach:

  • Sequence analysis of truA across Agrobacterium species with different host ranges

  • Expression profiling of truA under different environmental conditions relevant to grapevine colonization

  • Functional complementation experiments between A. vitis truA and homologs from other species

• Evolutionary analysis:

  • Phylogenetic analysis of truA in relation to host specialization

  • Analysis of selection pressure on truA sequences

  • Identification of A. vitis-specific structural features through homology modeling

This research direction connects to the known host-specific nature of A. vitis, which causes crown gall specifically in grapevines and demonstrates strain-specific biological control properties .

How can researchers differentiate between direct and indirect effects when studying truA mutants in A. vitis?

When analyzing phenotypes of truA mutants, several approaches can help distinguish direct from indirect effects:

• Complementation strategies:

  • Wild-type gene reintroduction

  • Controlled expression using inducible promoters

  • Cross-species complementation with well-characterized truA homologs

• Experimental controls:

  • Use of catalytically inactive mutants (e.g., D60A equivalent) that maintain RNA binding

  • Time-course experiments to distinguish primary from secondary effects

  • Parallel analysis of multiple independent mutant lines

• Multi-omics approaches:

  • Integrated transcriptomics, proteomics, and tRNA modification profiling

  • Ribosome profiling to assess translation effects

  • Metabolomics to identify downstream metabolic changes

• Specific phenotype quantification:

  • Growth rate measurements under various conditions

  • Virulence assays with standardized inoculation protocols

  • tRNA modification analysis using LC-MS techniques

The example from E. coli truA research, where complementation of clpA and clpP1 mutants showed dependence on vector copy number and timing of application, illustrates the complexity of interpreting such experiments .

What are common pitfalls in in vitro assays for tRNA modification by truA, and how can they be addressed?

Researchers should be aware of several technical challenges when assessing truA activity:

• Substrate preparation issues:

  • Inconsistent tRNA folding affecting accessibility of modification sites

  • Solution: Include refolding steps with controlled cooling rates and appropriate Mg²⁺ concentrations

  • Control: Include positive control reactions with well-characterized tRNA substrates

• Enzyme stability concerns:

  • Loss of activity during purification or storage

  • Solution: Optimize buffer conditions (glycerol, reducing agents)

  • Control: Regular activity checks with standardized assays

• Assay sensitivity limitations:

  • Difficulty detecting low levels of pseudouridylation

  • Solution: Employ multiple detection methods with different sensitivity ranges

  • Optimization: Determine optimal enzyme:substrate ratios and reaction conditions

• Artifact identification:

  • Non-enzymatic RNA degradation mistaken for activity

  • Control: Include no-enzyme controls and heat-inactivated enzyme controls

  • Validation: Confirm modification by orthogonal methods (mass spectrometry)

• Data analysis considerations:

  • Proper normalization for quantitative comparisons

  • Time-course experiments to determine reaction kinetics

  • Statistical analysis of replicate experiments

Experience from E. coli truA studies suggests that crystallization of enzyme-tRNA complexes may require testing multiple tRNA substrates, as some combinations may not yield crystals (as seen with tRNA leu2) .

How might CRISPR-Cas9 techniques be applied to study truA function in the context of A. vitis-grapevine interactions?

CRISPR-Cas9 technology offers powerful approaches for truA research:

• Genome editing applications:

  • Creation of precise truA mutants in A. vitis

    • Catalytic site mutations

    • Domain-specific mutations

    • Regulatory element modifications

  • Tagged variants for in vivo localization and interactome studies

  • Conditional expression systems for temporal control

• CRISPR interference (CRISPRi) approaches:

  • Tunable repression of truA expression

  • Study of dosage effects without complete gene deletion

  • Tissue or condition-specific knockdown during plant infection

• Base editing applications:

  • Introduction of specific truA mutations without double-strand breaks

  • Targeted modification of tRNA substrates to probe truA specificity

  • Engineering of truA variants with altered substrate specificity

• Experimental design considerations:

  • Efficient delivery methods for CRISPR components into A. vitis

  • Off-target analysis specific to A. vitis genome

  • Phenotypic screening approaches that capture subtle effects on virulence

These techniques would complement traditional approaches and could accelerate understanding of truA's role in A. vitis biology and pathogenesis.

What computational approaches could predict the impact of specific truA mutations on tRNA recognition and modification in A. vitis?

Computational methods offer valuable predictive power for truA research:

• Structural modeling approaches:

  • Homology modeling of A. vitis truA based on E. coli structures

  • Molecular docking of A. vitis tRNAs to the modeled enzyme

  • Molecular dynamics simulations to capture enzyme-substrate interactions

• Machine learning applications:

  • Prediction of pseudouridylation sites in tRNAs

  • Classification of tRNAs by likely modification efficiency

  • Feature extraction to identify key determinants of truA recognition

• Phylogenetic and evolutionary analyses:

  • Identification of conserved versus variable regions across bacterial truA orthologs

  • Correlation of truA sequence features with host specificity

  • Detection of selection signatures associated with host adaptation

• Network analysis:

  • Prediction of truA's position in A. vitis gene regulatory networks

  • Identification of potential functional partners based on co-expression data

  • Integration with systems biology models of A. vitis pathogenesis

These computational approaches can generate testable hypotheses and guide the design of targeted experiments, complementing traditional biochemical and genetic studies.