Recombinant Desulfovibrio vulgaris Queuine tRNA-ribosyltransferase (tgt)

What is Queuine tRNA-ribosyltransferase (TGT) and what is its primary biological role?

Queuine tRNA-ribosyltransferase (TGT), also known as tRNA-guanine transglycosylase, is an enzyme that catalyzes the exchange of guanine (G) with queuine (Q) at position 34 of specific tRNAs, resulting in hypermodified transfer RNAs . This post-transcriptional modification is essential for proper protein synthesis and cellular signaling networks.

The enzyme functions by precisely removing the genetically encoded guanine at the wobble position (position 34) of tRNAs and replacing it with queuine or its precursor, depending on the organism. In bacteria, TGT typically incorporates preQ₁ (a queuine precursor), while eukaryotic TGT directly incorporates queuine .

The modification at this crucial position affects codon recognition and translational efficiency, making TGT an important component of the cellular machinery that regulates gene expression at the translational level.

How does bacterial TGT structurally differ from eukaryotic TGT, and what implications does this have for research?

Bacterial and eukaryotic TGTs differ significantly in their quaternary structure:

• Bacterial TGT: Functions as a homodimer, with both subunits being catalytically active .

• Eukaryotic TGT: Functions as a heterodimer comprised of:

  • A catalytic subunit (QTRT1/TGT)

  • A non-catalytic subunit (QTRT2) that plays a crucial role in tRNA binding

A landmark 2023 cryo-EM structure of human TGT revealed that the non-catalytic subunit in eukaryotic TGT contains a two-stranded βEβF-sheet that serves as an additional RNA-binding motif, critical for stable interaction with the complete tRNA molecule . This structural feature is absent in bacterial homologs.

For researchers working with Desulfovibrio vulgaris TGT, this distinction is important when designing experiments, as bacterial TGT's homodimeric structure means that experimental approaches will differ from those used with eukaryotic enzymes. Proper experimental design should account for these structural differences, especially when studying substrate binding or developing inhibitors.

Which specific tRNAs are substrates for TGT modification and what is the significance of these modifications?

TGT specifically modifies four tRNA isoacceptors:

• tRNA^Asp

• tRNA^Asn

• tRNA^His

• tRNA^Tyr

These are the isoacceptors responsible for decoding NAC and NAU codons . The queuosine modification at the wobble position (position 34) of these tRNAs has significant effects on translation:

• It regulates translational speed by inverting a tRNA's preference for C- or U-ending synonymous codons, though the direction of this preference depends on both species and codon type .

• In pathogenic bacteria, such as Shigella flexneri, queuosine deficiency leads to decreased virulence, potentially because certain virulence factor mRNAs are themselves Q-modified .

• The modification affects the accuracy and efficiency of translation, particularly at the wobble position, which is crucial for proper decoding of the genetic code.

For researchers studying Desulfovibrio vulgaris TGT, understanding these substrate specificities is essential for designing appropriate experimental assays and interpreting results in a physiological context.

What are the most effective methods for expressing and purifying recombinant TGT for structural and functional studies?

Expression Systems:

• Bacterial expression: E. coli BL21(DE3) with pET-based vectors is commonly used for bacterial TGTs

• Parameters to optimize:

  • Induction temperature (typically 18-25°C for improved solubility)

  • IPTG concentration (0.1-0.5 mM)

  • Expression duration (4-16 hours)

Purification Protocol:

• Lysis: Sonication or French press in buffer containing:

  • 50 mM Tris-HCl, pH 7.5-8.0

  • 300 mM NaCl

  • 5-10% glycerol

  • 1 mM DTT or 2-mercaptoethanol

  • Protease inhibitors

• Chromatography sequence:

  • Affinity chromatography (Ni-NTA for His-tagged constructs)

  • Ion exchange chromatography (typically Q-Sepharose)

  • Size exclusion chromatography (Superdex 200)

• Storage conditions:

  • Buffer containing 20 mM Tris-HCl, pH 7.5, 100 mM NaCl, 5% glycerol

  • Flash-freeze in liquid nitrogen and store at -80°C

For optimal results with Desulfovibrio vulgaris TGT specifically, researchers may need to modify these conditions. Mobilization strategies using conjugation, similar to those employed with Desulfovibrio desulfuricans, may be necessary if transformation efficiency is low .

How is TGT enzyme activity accurately measured in experimental settings?

TGT activity can be measured using several complementary approaches:

1. Base-exchange assay:

• Monitor the incorporation of radiolabeled guanine/preQ₁/queuine into tRNA substrates

• Initial velocities are determined by converting the slopes of time-course plots

• Results are typically analyzed using Michaelis-Menten kinetics

2. Kinetic parameters determination:
Kinetic experiments should measure:

• k_cat (catalytic rate constant)

• K_m (Michaelis constant for substrate binding)

• k_cat/K_m (catalytic efficiency)

Sample kinetic data for wild-type and mutant TGT enzymes:

Standard errors shown in parentheses. Parameters calculated from three replicate determinations .

3. HPLC analysis:

• Separation and quantification of modified nucleosides after tRNA digestion

• Allows for detection of queuosine incorporation without radioactive labels

When studying Desulfovibrio vulgaris TGT, researchers should optimize these methods based on the specific biochemical properties of this enzyme.

What structural determination techniques have provided the most valuable insights into TGT architecture?

Multiple structural biology techniques have contributed to our understanding of TGT architecture:

1. X-ray crystallography:

• Historically provided high-resolution structures of TGT in complex with substrate analogs

• Revealed active site architecture and substrate binding modes

• Limited in providing information about dynamic regions and complex formation with full-length tRNA

2. Cryo-electron microscopy (cryo-EM):

• Recent breakthrough: 2023 cryo-EM structure of human TGT in complex with complete tRNA

• Revealed previously unknown tRNA binding interactions, particularly with the non-catalytic subunit

• Provided insights into conformational changes upon tRNA binding

3. Small-angle X-ray scattering (SAXS):

• Used to study solution state conformation and flexibility

• Complementary to crystallography for analyzing conformational ensembles

• Helps identify disordered regions and conformational transitions

4. UV-crosslinking:

• Identified additional RNA-binding motifs in the non-catalytic subunit (QTRT2) of eukaryotic TGT

• Used in combination with mutagenesis to map interaction surfaces

For researchers studying Desulfovibrio vulgaris TGT, a combination of these methods would provide the most comprehensive structural characterization, beginning with crystallography for high-resolution details of the active site and progressing to techniques that capture dynamics and larger complex formations.

How do specific active site mutations affect TGT catalytic efficiency and substrate specificity?

Mutagenesis studies of TGT have provided crucial insights into structure-function relationships. The table below shows how mutations at position Cys145 affect kinetic parameters:

Standard errors shown in parentheses. Parameters calculated from three replicate determinations .

Key findings from these data:

• The Cys145Ala mutation dramatically increases k_cat by ~15-fold, suggesting that the wild-type cysteine may restrict catalytic rate

• The Cys145Asp mutation severely impairs catalytic efficiency (15-fold decrease), likely due to charge repulsion with the substrate

• The Cys145Ser mutation represents an intermediate case, with improved catalysis but modestly increased K_m

These findings demonstrate how single amino acid substitutions can significantly alter both substrate binding (K_m) and catalytic turnover (k_cat). For researchers working with Desulfovibrio vulgaris TGT, similar mutagenesis studies would be valuable for understanding species-specific catalytic mechanisms.

What is the connection between TGT expression and human disease, particularly cancer?

Emerging evidence suggests important connections between TGT/QTRT1 and cancer:

• Lung Adenocarcinoma (LUAD):

  • Enhanced expression of QTRT1 has been observed in LUAD compared to normal tissue

  • Western blotting has shown that QTRT1 expression in the mitochondria of human LUAD A549 cells is higher than in normal human bronchial epithelial 16HBE cells

  • Immunohistochemistry (IHC) results confirm significantly higher positive expression of QTRT1 in LUAD compared to normal lung tissues

• Colon Adenocarcinoma:

  • QTRT1 may be involved in the carcinogenesis of human colon adenocarcinoma through modulation of tRNAs

  • Specific tRNAs related to human colon adenocarcinoma have been identified

• Breast Cancer:

  • Specific tRNAs modified by TGT have been related to human breast cancer

Researchers have explored the relationship between QTRT1 expression, methylation, and clinical features (including demographic factors, pathology stage, residual tumor, and survival outcome) in LUAD using gene expression data from 1,012 LUAD samples and 112 normal controls .

For researchers studying bacterial TGTs like that from Desulfovibrio vulgaris, these connections highlight the evolutionary importance of queuosine modification and suggest potential for comparative studies between bacterial and human systems.

How can computational approaches enhance our understanding of TGT function and substrate recognition?

Computational methods provide valuable insights into TGT function that complement experimental approaches:

1. Molecular Dynamics (MD) Simulations:

• Reveal conformational changes during substrate binding and catalysis

• Identify water molecules and metal ions crucial for catalysis

• Typical protocol: 100-500 ns simulations in explicit solvent with AMBER or CHARMM force fields

2. Quantum Mechanics/Molecular Mechanics (QM/MM):

• Essential for understanding the chemical reaction mechanism

• Allows modeling of bond breaking/formation during base exchange

• Typically uses DFT methods for the QM region (active site) and classical force fields for the rest of the protein

3. Homology Modeling:

• Useful for predicting structures of TGTs from species lacking experimental structures

• For Desulfovibrio vulgaris TGT, homology modeling based on other bacterial TGTs would be a valuable starting point

• Requires careful template selection (30-40% sequence identity minimum) and model validation

4. Molecular Docking:

• Predicts binding modes of substrates and potential inhibitors

• Should be validated against experimental binding data

• Can guide the design of species-selective inhibitors

5. Sequence and Structural Bioinformatics:

• Multiple sequence alignments reveal conserved catalytic residues

• Structural alignments identify common binding motifs

• Analysis of co-evolving residues can identify functionally linked positions

These computational approaches are particularly valuable for studying Desulfovibrio vulgaris TGT when experimental data is limited, as they can generate testable hypotheses about enzyme function based on comparisons with better-characterized TGTs.

What is the significance of the non-catalytic subunit in eukaryotic TGT and what are the implications for evolutionary studies?

The non-catalytic subunit of eukaryotic TGT (QTRT2) has emerged as a crucial component with significant evolutionary implications:

• Structural role: The 2023 cryo-EM structure revealed that the non-catalytic subunit plays a critical role in tRNA binding, with the two-stranded βEβF-sheet serving as an additional RNA-binding motif .

• Functional significance:

  • UV-crosslinking and mutagenesis experiments have identified specific regions of QTRT2 that interact with tRNA

  • These additional tRNA-binding sites likely enhance substrate specificity and binding affinity

• Evolutionary considerations:

  • The transition from homodimeric bacterial TGT to heterodimeric eukaryotic TGT represents a significant evolutionary adaptation

  • The non-catalytic subunit may have evolved from a gene duplication event followed by specialization

  • Comparative analysis between bacterial TGTs (like Desulfovibrio vulgaris TGT) and eukaryotic heterodimeric TGTs provides insights into the evolution of enzyme complexity

• Solution state dynamics:

  • Analysis of solution state conformation, flexibility, and disorder of apo TGT has revealed conformational transitions upon tRNA binding

  • These dynamics appear to be regulated differently in the bacterial versus eukaryotic systems

For researchers studying Desulfovibrio vulgaris TGT, understanding the homodimeric nature of bacterial TGT in contrast to the heterodimeric eukaryotic enzyme provides context for interpreting experimental results and may guide approaches to engineer enhanced or altered activity.

How does queuosine modification affect translation and cellular physiology?

Queuosine modification at the wobble position of tRNAs has multifaceted effects on cellular physiology:

• Translational regulation:

  • Regulates translational speed by altering tRNA preference for C- or U-ending synonymous codons

  • The direction of codon preference is species and codon-type dependent

  • Affects reading frame maintenance and translational accuracy

• Cellular signaling:

  • tRNAs are central components of protein synthesis and cell signaling networks

  • Modulation of these tRNAs through queuosine modification can specifically impact cancer progression

• Stress response:

  • Queuosine modification levels change under various stress conditions

  • May represent an adaptive mechanism to modulate the proteome under stress

• Bacterial pathogenesis:

  • In Shigella flexneri, queuosine deficiency leads to loss of virulence

  • This effect may be mediated through Q-modification of virF mRNA

For researchers studying Desulfovibrio vulgaris TGT, investigating how queuosine modification affects the organism's adaptation to its unique anaerobic environment could provide novel insights into the physiological role of this modification in diverse bacterial species.

What methodological approaches are recommended for studying TGT in the context of a complete cellular system?

To understand TGT function in a complete cellular context, researchers should consider these methodological approaches:

1. Gene knockout/knockdown studies:

• CRISPR-Cas9 gene editing in model organisms

• RNAi knockdown to create partial loss of function

• Phenotypic analysis of growth, stress response, and metabolic profiles

2. Metabolomics analysis:

• Liquid chromatography-mass spectrometry (LC-MS) to profile tRNA modifications

• Quantification of queuosine and its precursors in different cellular conditions

• Comparative analysis between wild-type and TGT-deficient strains

3. Ribosome profiling:

• Analysis of translational efficiency at codons read by Q-modified tRNAs

• Identification of genes most affected by absence of queuosine modification

• Protocol typically includes:

  • Cycloheximide treatment to freeze ribosomes

  • RNase digestion to generate ribosome-protected fragments

  • Deep sequencing and computational analysis

4. Proteomics approaches:

• Stable isotope labeling by amino acids in cell culture (SILAC)

• Pulsed SILAC to measure protein synthesis rates

• Analysis of proteome changes in TGT-deficient cells

5. Systems biology integration:

• Network analysis incorporating transcriptomics, proteomics, and metabolomics data

• Computational modeling of translation with and without Q-modification

• Flux balance analysis to predict metabolic consequences

For studying Desulfovibrio vulgaris TGT specifically, these approaches should be adapted to anaerobic culturing conditions and the specific metabolic pathways of this organism. Collaboration between microbiologists, biochemists, and computational biologists would yield the most comprehensive understanding of TGT function in cellular context.