Recombinant Staphylococcus aureus Queuine tRNA-ribosyltransferase (tgt)

What is Staphylococcus aureus Queuine tRNA-ribosyltransferase (tgt)?

Staphylococcus aureus Queuine tRNA-ribosyltransferase (tgt) is an enzyme that catalyzes the exchange of guanine (G) with queuine (Q) at position 34 in specific tRNAs. This enzyme (EC 2.4.2.29) belongs to the family of glycosyltransferases, specifically the pentosyltransferases . The recombinant form from S. aureus strain USA300/TCH1516 consists of 379 amino acids with a molecular function centered on creating hypermodified transfer RNAs that play crucial roles in protein synthesis and cell signaling networks .

What biochemical reaction does queuine tRNA-ribosyltransferase catalyze?

Queuine tRNA-ribosyltransferase catalyzes a specific exchange reaction:

[tRNA]-guanine + queuine → [tRNA]-queuine + guanine

This enzyme recognizes tRNAs with GUN anticodons corresponding to the amino acids histidine, aspartic acid, asparagine, and tyrosine. The enzyme removes guanine at the wobble position (position 34) and replaces it with queuine, resulting in hypermodified tRNAs. The systematic name for this enzyme is [tRNA]-guanine:queuine tRNA-D-ribosyltransferase, though it is also known by other names including tRNA-guanine transglycosylase, guanine insertion enzyme, Q-insertase, and others .

What are the optimal storage conditions for recombinant S. aureus tgt?

Recombinant S. aureus tgt requires specific storage conditions to maintain stability and activity:

• Short-term storage: -20°C

• Extended storage: -20°C to -80°C

• Working aliquots: 4°C for up to one week

For reconstitution, the vial should be briefly centrifuged before opening. The protein should be reconstituted in deionized sterile water to a concentration of 0.1-1.0 mg/mL. For long-term storage, adding glycerol to a final concentration of 5-50% (typically 50%) is recommended before creating aliquots for storage at -20°C/-80°C . This prevents protein degradation from repeated freeze-thaw cycles.

What methodological approaches can researchers use to assess tgt activity?

Several complementary methodologies can be employed to analyze tgt enzymatic activity:

• Radioisotope incorporation assays:

  • Incubating the enzyme with radiolabeled queuine and substrate tRNAs

  • Measuring incorporated radioactivity after precipitation and washing steps

  • Quantifying exchange activity via scintillation counting

• HPLC analysis of modified nucleosides:

  • Enzymatic digestion of tRNAs to nucleosides after modification

  • Chromatographic separation of modified versus unmodified nucleosides

  • UV detection and quantification of modification levels

• Mass spectrometry-based detection:

  • LC-MS/MS analysis of digested tRNAs

  • Precise identification and quantification of modified nucleosides

  • Determination of modification stoichiometry

• Kinetic parameter determination:

  • Time-course analysis of substrate conversion

  • Variation of substrate concentrations to determine Km and Vmax

  • Investigation of potential inhibitors via competitive inhibition studies

These methodologies allow researchers to comprehensively characterize the activity profile of recombinant S. aureus tgt under various experimental conditions.

How does queuosinylation affect translational dynamics and accuracy?

Queuosinylation has complex effects on translation that vary between organisms and codon contexts, as demonstrated by ribosome profiling studies:

In Schizosaccharomyces pombe, Q modification enhances translational efficiency of C-ending codons (CAC for histidine and GAC for aspartic acid) while decreasing the efficiency of U-ending codons (UAU for tyrosine and AAU for asparagine) . This creates a context-dependent effect on translational speed across different codons.

In S. pombe, absence of Q modification resulted in reduced translation of genes with mitochondrial function, leading to respiratory defects . This demonstrates that tRNA modification by tgt has genome-wide effects on specific gene classes and physiological functions.

What comparative differences exist between bacterial and eukaryotic tgt enzymes?

Several critical differences exist between bacterial tgt enzymes (like S. aureus tgt) and eukaryotic queuine tRNA-ribosyltransferases:

• Structural organization:

  • Bacterial tgt: Functions as a monomeric enzyme

  • Eukaryotic tgt: Functions as a heterodimer composed of QTRT1 (catalytic subunit) and QTRT2 (non-catalytic subunit)

• Substrate specificity:

  • Bacterial tgt: Can utilize preQ₁ (7-aminomethyl-7-deazaguanine, a queuine precursor) as substrate

  • Eukaryotic tgt: Specifically requires queuine as substrate

• Cellular processes:

  • Bacterial systems: Synthesize queuine precursors de novo

  • Eukaryotic systems: Cannot synthesize queuine and must obtain it from diet or gut microbiota

• Subcellular localization:

  • Bacterial tgt: Cytoplasmic

  • Eukaryotic tgt: Found in both cytoplasm and mitochondria with QTRT1 expression observed in mitochondria of human lung adenocarcinoma cells

These differences make bacterial tgt enzymes potential targets for antimicrobial development with minimal cross-reactivity to human enzymes.

How can researchers design effective experimental controls when working with recombinant tgt?

Rigorous experimental design for recombinant tgt studies should include these methodological controls:

• Enzyme activity controls:

  • Positive control: Known substrate with established modification kinetics

  • Negative control: Heat-inactivated enzyme preparation

  • Substrate specificity control: Non-cognate tRNAs not recognized by tgt

• Protein quality controls:

  • Purity assessment: SDS-PAGE analysis (>85% purity expected)

  • Activity verification: Standardized assay before experimental use

  • Stability control: Freshly thawed versus repeatedly frozen samples

• Reaction condition controls:

  • Buffer composition: Testing with and without essential cofactors

  • Temperature dependence: Activity assessment at varying temperatures

  • pH series: Determination of optimal pH for activity

• Specificity controls for tRNA modification:

  • Position-specific controls: tRNAs with position 34 mutations

  • Anticodon-stem loop mutants: Testing recognition requirements

  • Pre-modified substrates: tRNAs already containing queuine at position 34

• Methodological validation:

  • Multiple detection methods: Confirming results with orthogonal approaches

  • Technical replicates: Minimizing measurement error

  • Biological replicates: Assessing preparation-to-preparation variation

Implementing these controls ensures experimental rigor and facilitates accurate interpretation of results when studying recombinant S. aureus tgt.

How might tgt activity contribute to S. aureus pathogenicity and virulence?

The potential role of tgt in S. aureus pathogenicity represents an important research frontier, with several mechanistic hypotheses:

• Translational regulation of virulence factors:

  • tRNA modifications could influence translation efficiency of virulence genes

  • Codon bias in virulence factors may depend on modified tRNAs for optimal expression

  • Context-dependent translational effects might regulate virulence factor production temporally

• Adaptation to host environments:

  • Queuosinylation patterns may change in response to host-imposed stresses

  • Modified tRNAs could enhance bacterial survival under oxidative stress, nutrient limitation, or pH changes encountered during infection

  • Differential tgt activity might contribute to adaptation within specific host niches

• Biofilm formation and persistence:

  • tRNA modifications may influence expression of genes involved in biofilm development

  • Queuine modification in S. pombe affects genes with mitochondrial function and causes respiratory defects , suggesting potential metabolic roles relevant to biofilm formation

  • Persistence mechanisms might be linked to translational reprogramming through tRNA modification

• Host-pathogen interactions:

  • S. aureus tgt might influence expression of immune evasion factors

  • Translation of secreted toxins and immune modulators could be regulated by tRNA modification patterns

Research methodologies to investigate these connections include genetic knockouts of tgt in S. aureus, complementation studies, animal infection models, and comparative transcriptomic/proteomic analysis under infection-relevant conditions.

What structural approaches can reveal the mechanism of substrate recognition by tgt?

Multiple structural biology approaches can elucidate tgt substrate recognition mechanisms:

• X-ray crystallography:

  • Co-crystallization of S. aureus tgt with substrate tRNAs or substrate analogs

  • Structure determination at high resolution to identify contact residues

  • Analysis of catalytic site architecture and conformational changes upon substrate binding

• Cryo-electron microscopy (cryo-EM):

  • Visualization of tgt-tRNA complexes at near-atomic resolution

  • Capturing different states of the enzyme during catalysis

  • Analysis of large-scale conformational changes upon substrate binding

• Site-directed mutagenesis coupled with activity assays:

  • Systematic mutation of predicted key residues based on sequence conservation

  • Kinetic characterization of mutant enzymes to identify essential residues

  • Thermodynamic binding studies to quantify effects on substrate affinity

• Molecular dynamics simulations:

  • In silico modeling of enzyme-substrate interactions

  • Prediction of binding energy contributions from specific residues

  • Simulation of conformational changes during catalysis

• Hydrogen/deuterium exchange mass spectrometry (HDX-MS):

  • Identification of regions that undergo conformational changes upon substrate binding

  • Mapping of protein dynamics relevant to catalysis and substrate recognition

These complementary approaches would provide comprehensive insights into how S. aureus tgt recognizes and modifies its tRNA substrates, essential knowledge for understanding its function and for rational inhibitor design.

How do translational effects of tgt vary between different model organisms?

Translational effects of queuosinylation show species-specific patterns:

• Schizosaccharomyces pombe:

  • Q modification increases translational speed of C-ending codons (CAC, GAC)

  • Q modification decreases translational speed of U-ending codons (UAU, AAU)

  • Q modification suppresses misreading of the near-cognate GGC glycine codon

  • Lack of Q modification causes reduced translation of mitochondrial function genes

• Drosophila species:

  • Evolutionary preference for C-ending codons at conserved Q codon sites

  • This pattern suggests selective pressure for optimal translation at these positions

• Human cells:

  • Three U-ending codons were more strongly affected by absence of Q than respective C-ending codons

  • This differs from the pattern observed in S. pombe, suggesting species-specific effects

• QTRT1 in cancer cells:

  • Enhanced expression observed in lung adenocarcinoma compared to normal tissue

  • Possible role in cancer progression through modulation of specific tRNAs

These differences highlight that tgt function and the consequences of queuosinylation have evolved differently across species, with implications for how we interpret model organism data when studying tRNA modifications.

What evidence suggests tgt as a potential antimicrobial target?

Several lines of evidence support investigating S. aureus tgt as an antimicrobial target:

• Essential nature of tRNA modifications:

  • tRNA modifications are crucial for translational accuracy and efficiency

  • Disruption of these modifications can impact bacterial fitness and virulence

• Structural differences from human enzyme:

  • Bacterial tgt differs significantly from the eukaryotic heterodimeric enzyme

  • These differences offer selectivity potential for inhibitor development

  • S. aureus tgt is a monomeric enzyme while human requires QTRT1/QTRT2 complex

• Potential virulence connection:

  • tRNA modifications can influence expression of virulence factors

  • Targeting tgt might reduce pathogenicity without directly killing bacteria (anti-virulence approach)

• Novel mechanism of action:

  • tgt inhibitors would represent a new class of antibiotics

  • Novel targets are critically needed to address antimicrobial resistance crisis

• Conserved across pathogenic species:

  • tgt is present in many bacterial pathogens

  • Potential for broad-spectrum applications while maintaining selectivity against human enzyme

Research approaches for antimicrobial development include structure-based design of competitive inhibitors, high-throughput screening, and in vivo efficacy testing in animal infection models.

What methodological approaches can assess the impact of tgt inhibition on bacterial phenotypes?

Multiple experimental approaches can evaluate the phenotypic consequences of tgt inhibition:

• Genetic manipulation strategies:

  • Gene knockout or knockdown to create tgt-deficient strains

  • Conditional expression systems to study essentiality

  • Point mutations to create catalytically impaired variants

  • Complementation studies to confirm phenotype specificity

• Growth and fitness assessments:

  • Growth curve analysis under various nutrient conditions

  • Competition assays between wild-type and tgt-deficient strains

  • Stress resistance testing (oxidative, acid, heat shock)

  • Long-term evolution experiments to detect compensatory adaptations

• Virulence-related phenotype analysis:

  • Biofilm formation quantification

  • Hemolytic activity measurement

  • Host cell adhesion and invasion assays

  • Toxin production assessment

• Molecular analysis of tRNA modification status:

  • LC-MS/MS analysis of tRNA modification profiles

  • Northern blotting to detect changes in tRNA abundance

  • Ribosome profiling to assess translation efficiency changes

  • RNA sequencing to identify compensatory transcriptional responses

• In vivo relevance:

  • Animal infection models with tgt-deficient strains

  • Comparative virulence assessment

  • In vivo competition assays

  • Host immune response characterization

These methodological approaches provide a comprehensive framework for evaluating tgt as a potential antimicrobial target in S. aureus.