Recombinant Natranaerobius thermophilus Queuine tRNA-ribosyltransferase (tgt)

What is Queuine tRNA-ribosyltransferase (tgt) and what is its primary function?

Queuine tRNA-ribosyltransferase (tgt, EC 2.4.2.29) is an enzyme responsible for the post-transcriptional modification of specific tRNAs. This enzyme catalyzes the exchange of guanine at position 34 (the wobble position of the anticodon) with queuine, resulting in queuosine (Q) modification in tRNAs . The enzyme is also known as guanine insertion enzyme or tRNA-guanine transglycosylase according to its protein nomenclature .

The primary function of tgt is critical for fine-tuning protein translation. The Q modification of tRNAs modulates the translation rate of NAU codons by preventing translational codon bias shown by unmodified tRNAs and potentially increasing translational efficiency . This represents an important mechanism for regulating gene expression at the translational level with significant downstream effects on various cellular processes including biofilm formation and virulence in bacteria .

Which specific tRNAs are modified by tgt?

Tgt specifically modifies four types of tRNAs corresponding to amino acids with NAU codons:

• tRNA^Tyr (tyrosine)

• tRNA^His (histidine)

• tRNA^Asn (asparagine)

• tRNA^Asp (aspartic acid)

The modification occurs at the wobble anticodon position (position 34) of these tRNAs, where a guanine base is replaced with the modified queuosine (Q) base . This specific targeting pattern is highly conserved across diverse prokaryotic and eukaryotic species, suggesting the evolutionary importance of this modification . The presence of Q modification at this critical position affects how these tRNAs interact with their corresponding codons during protein synthesis, potentially altering the efficiency and accuracy of translation.

What distinguishes prokaryotic from eukaryotic tgt mechanisms?

The mechanisms of tgt function differ significantly between prokaryotes and eukaryotes:

In prokaryotes:

• Bacteria can synthesize Q-modified tRNA de novo through their own biosynthetic pathways

• The prokaryotic tgt functions as a single enzyme capable of catalyzing the entire reaction

• Bacterial tgt enzymes such as the one from Natranaerobius thermophilus are relatively well-characterized

In eukaryotes:

• Eukaryotic organisms cannot synthesize Q-modified tRNA de novo and depend on external sources of queuine

• Mammals strictly rely on gut microbiomes or diet to obtain the queuine base

• Eukaryotic tgt requires cooperation between two proteins

• In mouse studies, a homologue of the E. coli TGT works in conjunction with a protein called Qv1 (QTRTD1 variant_1)

• Neither protein alone can complement a tgt mutation in E. coli, but together they exhibit transglycosylase activity in vitro

This functional divergence highlights the evolutionary complexity of this modification system and the interdependence between mammals and their microbiome for maintaining this essential tRNA modification.

How is queuosine modification distributed across different organisms?

Queuosine modification displays an interesting distribution pattern across the tree of life:

• The Q-modification is almost universal in eubacterial and eukaryotic species

• Only bacteria possess the complete biosynthetic pathway to synthesize Q-modified tRNA de novo

• Eukaryotes, including mammals, depend on external sources of queuine

• Mammals obtain queuine through gut microbiomes or diet, establishing a nutritional dependency

• The degree of Q-modification in tRNAs can vary based on physiological conditions and may be impaired in certain pathological states such as cancer cells

This distribution pattern suggests potential symbiotic relationships between mammals and their microbiota, where bacteria provide essential modified nucleosides that the hosts cannot synthesize themselves. Additionally, the near-universal presence of this modification across diverse organisms underscores its fundamental role in cellular function that has been conserved throughout evolution.

What experimental methods can be used to quantify queuosine modification levels in tRNAs?

Several experimental approaches can be employed to quantify queuosine modification levels in tRNAs:

• APB gel electrophoresis-based method:

  • This technique exploits the presence of a cis-diol in the Q modification

  • tRNAs with Q modifications migrate slower through polyacrylamide gels supplemented with N-acryloyl-3-aminophenylboronic acid (APB)

  • The difference in migration can be visualized by Northern blots using probes for specific tRNAs

  • This method allows for the quantification of modification levels in individual tRNAs

• Mass spectrometry-based approaches:

  • Liquid chromatography coupled with mass spectrometry (LC-MS) enables precise quantification of modified nucleosides

  • Requires isolation and enzymatic digestion of tRNA samples to individual nucleosides

  • Provides high sensitivity detection of modified bases and their relative abundance

• Reverse transcription-based methods:

  • Exploits the fact that some modifications, including Q, can cause reverse transcriptase to pause or introduce mutations

  • The resulting cDNA can be analyzed by sequencing to identify modification sites

  • Next-generation sequencing platforms can provide quantitative information across the transcriptome

Each of these methods has distinct advantages and limitations, making them suitable for different research contexts. The choice of method should be based on the specific research question, available equipment, and desired sensitivity level.

How does tRNA Q-modification affect translation efficiency and codon bias?

The Q-modification of tRNAs has significant effects on translation efficiency and codon bias:

Understanding these effects provides insight into how tRNA modifications serve as a sophisticated mechanism for post-transcriptional regulation of gene expression with far-reaching consequences for cellular physiology.

What role do Q-modified tRNAs play in bacterial biofilm formation and virulence?

Q-modified tRNAs have emerged as important regulators of bacterial biofilm formation and virulence:

• Enrichment in relevant functions:

  • Bioinformatic analysis reveals that Q-genes (NAU codon-enriched genes affected by Q-modification) are widely enriched in functions related to biofilm formation and virulence in bacteria

  • This enrichment is particularly notable in human pathogens, suggesting clinical relevance

• Experimental verification:

  • Altering the degree of tRNA Q-modification significantly affects biofilm formation and virulence in different model bacteria

  • This effect has been observed in both Gram-positive bacteria (e.g., Bacillus subtilis) and Gram-negative bacteria (e.g., E. coli, Pseudomonas putida)

  • Genetic manipulation of Q-modification pathways results in measurable changes to bacterial phenotypes

• General regulatory mechanism:

  • This represents the first reported general mechanism controlling biofilm formation and virulence across diverse bacterial species

  • The mechanism works through coordinating the expression of functionally related genes enriched in NAU codons

  • This regulatory layer adds complexity to our understanding of bacterial physiology and pathogenesis

• Implications for pathogenicity:

  • Bioinformatic and experimental data suggest that Q enhances the virulence of most human pathogenic bacteria

  • This indicates potential therapeutic targets for addressing bacterial infections

  • Modulating Q-modification could represent a novel strategy for anti-virulence therapeutics

These findings highlight the importance of Q-modification in bacterial pathogenicity and suggest new approaches for understanding and potentially treating bacterial infections by targeting tRNA modification pathways.

How do environmental factors influence the activity of recombinant Natranaerobius thermophilus tgt?

The activity of recombinant Natranaerobius thermophilus tgt is influenced by various environmental factors that should be considered in experimental design:

• Temperature effects:

  • N. thermophilus is a thermophilic organism, suggesting its tgt likely has optimal activity at elevated temperatures

  • Temperature stability is a key consideration for maintaining enzyme activity during extended reactions

  • Temperature optimization may reveal activity profiles distinct from mesophilic tgt enzymes

• pH and buffer conditions:

  • Optimal pH range for enzyme activity must be determined empirically

  • Buffer composition can significantly affect enzyme stability and function

  • Compatibility with co-factors and substrates must be considered

• Salt concentration:

  • N. thermophilus is a halophilic organism adapted to high salt environments

  • The enzyme may require specific ionic conditions for optimal activity

  • Salt type and concentration may affect substrate binding and catalytic efficiency

• Storage conditions:

  • Recommended storage at -20°C, with extended storage at -20°C or -80°C

  • Repeated freezing and thawing is not recommended as it can denature the protein

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

• Reconstitution parameters:

  • Reconstitution in deionized sterile water to a concentration of 0.1-1.0 mg/mL

  • Addition of glycerol (final concentration 5-50%, with 50% as default) for long-term storage

  • Brief centrifugation prior to opening vials is recommended to collect contents

These environmental parameters must be systematically optimized to achieve maximal enzyme activity and stability, particularly when working with extremophilic enzymes that may have unusual optimal conditions compared to mesophilic counterparts.

What structural characteristics make Natranaerobius thermophilus tgt suitable for in vitro studies?

Natranaerobius thermophilus tgt possesses several structural characteristics that make it particularly valuable for in vitro studies:

• Thermostability properties:

  • As derived from a thermophilic organism, the enzyme exhibits high thermal stability

  • This property allows for experiments at elevated temperatures, potentially increasing reaction rates

  • Thermostability reduces concerns about denaturation during experimental procedures

• Protein structural features:

  • The full-length protein contains 370 amino acids with a complete sequence available

  • The protein sequence includes conserved domains essential for tgt catalytic function

  • The structure incorporates features that contribute to both thermostability and halotolerance

• Purity considerations:

  • The recombinant protein can be produced at >85% purity as determined by SDS-PAGE

  • This high purity minimizes interference from contaminating proteins in in vitro assays

  • Consistent purity levels enable reproducible experimental results

• Expression system compatibility:

  • Successfully expressed in E. coli expression systems

  • This indicates compatibility with standard prokaryotic expression systems used in laboratory settings

  • Potential for high-yield production of the recombinant enzyme

• Storage stability:

  • Relatively long shelf life (liquid form: 6 months at -20°C/-80°C; lyophilized form: 12 months at -20°C/-80°C)

  • This stability facilitates long-term experimental planning and reduces variability

  • Glycerol can be added as a cryoprotectant to further enhance stability

These characteristics make N. thermophilus tgt an excellent model enzyme for studying tRNA modification mechanisms, enzyme kinetics, and structure-function relationships in controlled laboratory conditions.

How can experimental conditions be optimized for tgt activity assays?

Optimizing experimental conditions for tgt activity assays requires consideration of multiple factors:

• Substrate parameters:

  • Selection of appropriate tRNA substrates (tRNA^Tyr, tRNA^His, tRNA^Asn, tRNA^Asp)

  • Substrate concentration optimization to avoid saturation or limitation effects

  • Assessment of substrate purity and integrity prior to assays

• Buffer optimization:

  • Systematic testing of pH values to determine optimal range

  • Inclusion of stabilizing agents like glycerol to maintain enzyme activity

  • Appropriate salt concentration, considering the halophilic nature of N. thermophilus

  • Testing different buffer systems (phosphate, Tris, HEPES) for compatibility

• Temperature parameters:

  • Determination of temperature optima through activity vs. temperature profiling

  • Evaluation of thermal stability over the assay duration

  • Potential for higher activity at elevated temperatures due to thermophilic origin

• Reaction monitoring strategies:

  • APB gel electrophoresis for detecting Q-modified vs. unmodified tRNAs

  • Mass spectrometry for quantitative analysis of modification levels

  • Radioactive labeling approaches for high sensitivity detection

• Kinetic parameter determination:

  • Time course experiments to determine linear range of reaction

  • Enzyme concentration optimization to achieve measurable rates

  • Substrate titration to determine Km and Vmax values

  • Inhibitor studies to characterize enzyme mechanism

• Quality control measures:

  • Inclusion of positive and negative controls in each experiment

  • Verification of enzyme activity prior to experimental use

  • Consistency checks across experimental replicates to ensure reproducibility

Systematic optimization of these parameters will ensure reliable and reproducible results in tgt activity assays, enabling more accurate characterization of enzyme properties and modification dynamics.

What expression systems are most effective for producing recombinant Natranaerobius thermophilus tgt?

Several expression systems can be employed for producing recombinant Natranaerobius thermophilus tgt, each with specific advantages and considerations:

• E. coli expression system:

  • The search results indicate that N. thermophilus tgt has been successfully expressed in E. coli

  • Advantages include rapid growth, high protein yields, and well-established protocols

  • Considerations include potential issues with protein folding and post-translational modifications

  • BL21(DE3) or similar strains are commonly used for recombinant protein expression

  • IPTG-inducible promoters offer controlled expression

• Alternative prokaryotic systems:

  • Bacillus subtilis may offer advantages for expressing proteins from Gram-positive bacteria

  • Specialized thermophilic expression hosts might improve folding of thermostable proteins

  • Considerations include different codon usage patterns and potential yield differences

• Expression optimization strategies:

  • Temperature adjustment: lower temperatures (15-25°C) often improve folding of recombinant proteins

  • Induction conditions: optimization of inducer concentration and induction timing

  • Media composition: enriched media for higher biomass and protein yields

  • Codon optimization: adapting the coding sequence to the preferred codons of the expression host

• Purification approach:

  • Addition of affinity tags (His-tag, GST, etc.) for simplified purification

  • Tag removal considerations if native protein is required for specific applications

  • Chromatography steps to achieve >85% purity as specified in the product information

The optimal expression system should be selected based on the specific requirements of the downstream applications, including protein yield, purity needs, and structural integrity considerations.

How can researchers verify the activity of recombinant tgt in experimental settings?

Verifying the activity of recombinant tgt in experimental settings can be accomplished through several complementary approaches:

• In vitro transglycosylase assay:

  • Using purified tRNAs as substrates for the enzyme

  • Measuring the replacement of guanine with queuine at position 34

  • Detection of modified tRNAs using APB gel electrophoresis and Northern blotting

  • Quantification of modification efficiency under controlled conditions

• Complementation assays:

  • Testing whether the recombinant tgt can restore function in tgt-deficient bacterial strains

  • Observing phenotypic changes related to Q-modification (e.g., biofilm formation)

  • Note that for eukaryotic-like tgt systems, individual components may not complement bacterial mutations, as seen with mouse TGT and Qv1

• Mass spectrometry-based verification:

  • Direct detection of queuosine-modified nucleosides in tRNA after enzyme treatment

  • Quantitative comparison of modification levels before and after enzyme addition

  • Identification of modification sites with nucleotide resolution

• Functional assays based on known Q-related phenotypes:

  • Assessing changes in bacterial biofilm formation after tgt treatment

  • Measuring virulence factor expression levels in model organisms

  • Testing NAU codon translation efficiency in appropriate systems

• Essential controls:

  • Heat-inactivated enzyme as negative control to confirm enzyme-dependent activity

  • Well-characterized tgt from model organisms (e.g., E. coli) as positive control

  • Substrate-free and enzyme-free reactions to rule out contamination or artifacts

These verification methods provide multiple lines of evidence for tgt activity, ensuring confidence in the functionality of the recombinant enzyme for subsequent experiments.

What are the optimal storage conditions for maintaining tgt stability and activity?

Maintaining the stability and activity of tgt requires careful attention to storage conditions:

• Temperature parameters:

  • Store at -20°C for regular use

  • For extended storage, conserve at -20°C or -80°C

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

  • Avoid repeated freezing and thawing which can lead to protein denaturation

• Solution preparation guidelines:

  • Briefly centrifuge vials prior to opening to bring contents to the bottom

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

  • Add glycerol to a final concentration of 5-50% (default recommendation: 50%) for cryoprotection

  • Filter sterilization may be considered for long-term storage solutions

• Aliquoting strategy:

  • Prepare small working aliquots to minimize freeze-thaw cycles

  • Use appropriate volume aliquots based on typical experimental needs

  • Label clearly with date, concentration, and buffer composition

• Expected shelf life:

  • Liquid form: approximately 6 months at -20°C/-80°C

  • Lyophilized form: approximately 12 months at -20°C/-80°C

  • Shelf life depends on multiple factors including buffer ingredients and storage temperature

• Quality control measures:

  • Periodically test enzyme activity to ensure functionality over time

  • Monitor for signs of precipitation or denaturation

  • Consider including stabilizing agents appropriate for thermophilic enzymes

Following these storage recommendations will help maximize the longevity and reliability of tgt preparations, ensuring consistent results across experimental timeframes.

How can tgt be used as a tool to study tRNA modification patterns in different cell types?

Tgt can serve as a powerful tool for studying tRNA modification patterns across different cell types:

• Comparative modification analysis:

  • Using recombinant tgt to assess the queuosine modification status of tRNAs from different cell types

  • Determining modification levels in normal vs. pathological states (e.g., cancer cells)

  • Comparing modification patterns across developmental stages or in response to environmental conditions

• Experimental approaches:

  • APB gel electrophoresis to separate Q-modified from unmodified tRNAs

  • Northern blotting with specific probes to identify individual tRNA species

  • Quantitative analysis of modification levels under different cellular conditions

  • Mass spectrometry to profile modification status across the tRNA pool

• Functional studies:

  • Using tgt to modify specific tRNAs in vitro followed by functional assays

  • Studying the impact of different modification levels on translation efficiency

  • Correlating tRNA modification status with expression of NAU codon-rich genes

  • Assessing phenotypic changes resulting from altered modification status

• Systems biology applications:

  • Investigating how tRNA modification patterns affect cellular proteomes

  • Examining relationships between microbiome composition and host tRNA modification status

  • Exploring links between tRNA modifications and specific cellular phenotypes

• Methodology workflow:

  • Isolate total tRNA from cell types of interest using appropriate extraction methods

  • Determine baseline Q-modification levels using APB gel electrophoresis

  • Treat unmodified or partially modified tRNAs with recombinant tgt

  • Analyze changes in modification patterns and correlate with cellular functions

This toolset enables researchers to uncover how tRNA modification patterns vary across biological contexts and contribute to cellular function and dysfunction in different tissues, disease states, and experimental conditions.

What analytical techniques can be combined with tgt-based assays to study translation dynamics?

Combining tgt-based assays with complementary analytical techniques provides comprehensive insights into translation dynamics:

• Ribosome profiling:

  • Assess ribosome positioning on mRNAs with different codon composition

  • Determine how Q-modification affects translation elongation rates at NAU codons

  • Compare ribosome occupancy patterns before and after modulating Q-tRNA levels

  • Identify potential ribosome pausing sites affected by Q-modification status

• Mass spectrometry-based proteomics:

  • Quantify protein expression changes resulting from altered Q-modification status

  • Identify specific proteins (encoded by Q-genes) most affected by tgt activity

  • Analyze post-translational modifications that might be influenced by translation rates

  • Perform temporal proteomics to capture dynamic changes in protein synthesis

• Computational codon usage analysis:

  • Identify genes enriched in NAU codons (Q-genes) across different organisms

  • Predict functional categories particularly dependent on Q-modification

  • Model the impact of Q-modification on translational dynamics

  • Perform comparative genomics of codon usage across species with different tgt systems

• Single-molecule translation studies:

  • Real-time monitoring of translation rates with Q-modified vs. unmodified tRNAs

  • Fluorescence-based approaches to track individual translation events

  • Investigation of translation fidelity and error rates under varying Q-modification conditions

• Transcriptome-wide structure analysis:

  • Examine how translation rates influenced by Q-modification affect mRNA structure

  • Study co-translational folding dynamics of nascent peptides

  • Investigate potential regulatory feedback between translation dynamics and transcription

• Integrated experimental design:

  • Use tgt to establish controlled levels of Q-modification in tRNAs

  • Apply multiple analytical approaches to the same biological system

  • Correlate molecular changes with functional outcomes (e.g., biofilm formation, virulence)

This multi-faceted approach allows researchers to build a comprehensive understanding of how tRNA modifications influence the complex process of translation and its downstream effects on cellular physiology across different biological systems and experimental conditions.