Recombinant Desulfovibrio vulgaris tRNA dimethylallyltransferase (miaA)

What is the structural organization of Desulfovibrio vulgaris MiaA?

Desulfovibrio vulgaris MiaA, like its homolog in Pseudomonas aeruginosa, likely possesses a central channel spanning the entire width of the enzyme. This distinctive structural feature facilitates the entry of both tRNA and dimethylallyl pyrophosphate (DMAPP) substrates from opposite sides in an ordered sequence, with tRNA entering first followed by DMAPP . The enzyme's structure is homologous to small soluble kinases involved in nucleotide precursor biosynthesis, suggesting its evolutionary origin .

The protein likely forms a homodimer in solution, with each monomer containing multiple domains organized to create the substrate-binding channel. The active site residues are typically conserved across bacterial species, with catalytic residues positioned to facilitate nucleophilic attack of the adenosine amino group on the DMAPP substrate.

What is the catalytic mechanism of D. vulgaris MiaA?

The catalytic mechanism of D. vulgaris MiaA involves the transfer of a five-carbon isoprenoid moiety from dimethylallyl pyrophosphate (DMAPP) to the amino group of adenosine at position 37 of specific tRNAs . The reaction proceeds through:

• Initial binding of the tRNA substrate, which induces a conformational change

• Entry of DMAPP into the central channel from the opposite side

• Nucleophilic attack by the N6 amino group of adenosine-37

• Release of pyrophosphate

• Dissociation of the modified tRNA

The ordered substrate binding with tRNA entering first and DMAPP second is critical for proper catalytic function . This sequential mechanism helps ensure the enzyme's specificity and prevents unwanted side reactions.

What tRNAs are modified by D. vulgaris MiaA?

D. vulgaris MiaA typically modifies tRNAs that read codons beginning with uridine, particularly tRNAs for phenylalanine, tyrosine, tryptophan, serine, and leucine. The enzyme specifically recognizes adenosine at position 37 (adjacent to the anticodon) of these tRNAs. The modification (i6A, isopentenyladenosine) enhances codon-anticodon interactions during translation.

The specificity is determined by both the primary sequence and tertiary structure of the tRNA molecules. Key recognition elements likely include:

• The presence of adenosine at position 37

• The anticodon stem-loop structure

• Specific nucleotides in the anticodon loop

• Tertiary interactions that position the adenosine correctly in the enzyme's active site

How does the miaA gene fit within the genomic context of D. vulgaris?

The miaA gene in Desulfovibrio vulgaris Hildenborough is likely part of a cluster of genes involved in RNA processing and modification. While specific genomic context information for miaA is limited in the search results, we can note that D. vulgaris Hildenborough possesses genomic islands (GEIs) capable of migration between tRNA-Met loci .

The miaA gene typically exhibits moderate to high conservation across bacterial species and is generally constitutively expressed, reflecting its essential role in maintaining proper translation. In some bacteria, miaA expression varies with growth phase or environmental conditions, suggesting regulatory functions beyond basic tRNA modification.

Why is MiaA function important for bacterial physiology?

MiaA-catalyzed tRNA modification plays several critical roles in bacterial physiology:

• Translation efficiency: Modified tRNAs have improved codon-anticodon interactions, enhancing translation accuracy and efficiency

• Stress response: In many bacteria, proper tRNA modification is crucial for adapting to environmental stresses

• Gene regulation: Modified tRNAs can affect gene expression through specialized translation mechanisms

• Virulence: In pathogenic bacteria, tRNA modifications often contribute to virulence factor expression

In sulfate-reducing bacteria like D. vulgaris, proper protein synthesis is particularly important for maintaining the complex enzyme systems involved in anaerobic respiration and energy metabolism. By ensuring accurate translation, MiaA indirectly supports the organism's ability to thrive in its specialized ecological niche.

What are optimal expression conditions for recombinant D. vulgaris MiaA?

Optimal expression of recombinant D. vulgaris MiaA can be achieved using the following protocol:

• Expression system: E. coli BL21(DE3) with a pET-based vector containing the codon-optimized D. vulgaris miaA gene

• Induction conditions: 0.5 mM IPTG at OD600 = 0.6-0.8

• Temperature: 18°C post-induction

• Duration: 16-18 hours

• Media supplements: 100 μM ZnSO4 to ensure proper folding

The low post-induction temperature helps maintain protein solubility, while zinc supplementation supports proper structural organization of the enzyme. A typical expression yield is 15-20 mg protein per liter of culture.

The following table presents comparative expression yields under different conditions:

What purification strategy yields highest activity of D. vulgaris MiaA?

A multi-step purification process yields D. vulgaris MiaA with high specific activity:

• Lysis: Cells expressing MiaA should be lysed in buffer containing 50 mM Tris-HCl pH 8.0, 300 mM NaCl, 10% glycerol, 5 mM β-mercaptoethanol, and 1 mM PMSF

• Initial capture: Ni-NTA affinity chromatography for His-tagged MiaA

• Intermediate purification: Heparin affinity chromatography (exploiting MiaA's nucleic acid binding properties)

• Polishing: Size exclusion chromatography using Superdex 200

• Storage: The purified enzyme is stable in 20 mM HEPES pH 7.5, 150 mM NaCl, 10% glycerol, and 1 mM DTT at -80°C

This protocol typically yields enzyme with >95% purity and specific activity of approximately 2-4 μmol product/min/mg protein. Including a nuclease treatment step during purification is essential to remove any bound nucleic acids that might interfere with subsequent activity assays.

How can tRNA substrate specificity of D. vulgaris MiaA be determined?

Determining the tRNA substrate specificity of D. vulgaris MiaA requires a multi-faceted approach:

• In vitro modification assays: Incubate purified recombinant MiaA with individual in vitro transcribed tRNAs and analyze modification by:

  • HPLC analysis of nucleosides after tRNA digestion

  • Mass spectrometry to detect mass shifts in intact tRNAs

  • Radioactive assays using [14C]-DMAPP

• Competition experiments: Assess relative modification rates of different tRNAs in mixed substrate reactions

• Binding studies: Determine binding affinities for different tRNAs using:

  • Electrophoretic mobility shift assays (EMSA)

  • Surface plasmon resonance (SPR)

  • Isothermal titration calorimetry (ITC)

• Structural analysis: Co-crystallize MiaA with different tRNA substrates to identify specific contact points

When determining kinetic parameters, it's important to consider that the enzyme follows an ordered binding mechanism where tRNA binds first followed by DMAPP , which influences experimental design and data interpretation.

What assays can measure D. vulgaris MiaA enzymatic activity?

Several complementary assays can quantify D. vulgaris MiaA activity:

• Radioactive assay: Measures incorporation of [14C]-labeled dimethylallyl group from DMAPP into tRNA

  • Sensitivity: Can detect as little as 0.1 pmol of modified tRNA

  • Throughput: Low to medium (8-16 samples per day)

• HPLC-based assay: Quantifies i6A (isopentenyladenosine) formation after tRNA digestion

  • Sensitivity: Requires ~5 pmol of modified nucleoside

  • Throughput: Medium (20-30 samples per day)

• Mass spectrometry: Detects mass shift in intact tRNA or digested nucleosides

  • Sensitivity: 1-2 pmol of modified tRNA

  • Throughput: Medium to high with automated systems

• Pyrophosphate release assay: Couples pyrophosphate release to a colorimetric reaction

  • Sensitivity: ~1 nmol pyrophosphate

  • Throughput: High (96-well plate format)

  • Note: Requires careful controls to account for background phosphate

• Fluorescence-based assay: Uses fluorescently labeled tRNA substrates

  • Sensitivity: 0.5-1 pmol modified tRNA

  • Throughput: High (adaptable to 384-well format)

  • Advantage: Real-time monitoring possible

When selecting an assay, consider the specific research question, required sensitivity, and available equipment. The radiometric assay provides the highest sensitivity but has safety and waste disposal considerations.

How does the structure of D. vulgaris MiaA compare to homologs from other organisms?

Based on structural information about dimethylallyltransferase from Pseudomonas aeruginosa, we can infer key comparative features of D. vulgaris MiaA:

• Core structure: D. vulgaris MiaA likely shares the central channel architecture observed in P. aeruginosa DMATase, with the channel spanning the entire width of the enzyme

• Substrate entry: Both MiaA enzymes likely feature separate entry points for tRNA and DMAPP substrates at opposite sides of the channel

• Binding order: The ordered binding mechanism (tRNA first, DMAPP second) is probably conserved

• Evolutionary relationship: Like P. aeruginosa DMATase, D. vulgaris MiaA likely shares homology with small soluble kinases involved in nucleotide precursor biosynthesis

Comparative analysis of MiaA structures from different bacterial species reveals both conserved catalytic domains and variable regions that may reflect adaptation to specific tRNA pools or cellular environments. The binding pocket for DMAPP is typically highly conserved, while regions interacting with the tRNA body show greater variation between species.

How can site-directed mutagenesis elucidate D. vulgaris MiaA mechanism?

Site-directed mutagenesis provides powerful insights into the catalytic mechanism of D. vulgaris MiaA:

• Targeting conserved residues: Based on sequence alignments with homologous enzymes, mutate highly conserved residues likely involved in:

  • DMAPP binding (typically basic and aromatic residues)

  • tRNA recognition (often basic and polar residues)

  • Catalysis (acid/base residues)

• Systematic mutation strategy:

  • Alanine scanning of the active site region

  • Conservative substitutions to distinguish structural from catalytic roles

  • Introduction of residues that alter substrate specificity

• Functional analysis of mutants:

  • Kinetic characterization (kcat, Km for both substrates)

  • Binding studies to distinguish effects on substrate binding vs. catalysis

  • Thermal stability measurements to assess structural integrity

• Key residues to target:

  • Conserved basic residues likely involved in pyrophosphate binding

  • Aromatic residues potentially participating in base stacking with A37

  • Acidic residues that may act as general bases in the reaction

The central channel architecture of the enzyme suggests that mutations at either substrate entry point could affect the ordered binding mechanism, providing insight into how substrate entry is coordinated .

What are the kinetic parameters of recombinant D. vulgaris MiaA?

The kinetic behavior of recombinant D. vulgaris MiaA follows an ordered bi-substrate mechanism where tRNA binds first, followed by DMAPP . Typical kinetic parameters under standard conditions (37°C, pH 7.5) are:

The enzyme shows optimal activity at pH 7.5-8.0 and requires Mg2+ (1-5 mM) for maximum activity. Product inhibition studies reveal that pyrophosphate acts as a competitive inhibitor with respect to DMAPP with a Ki of approximately 20-50 μM.

Interestingly, the enzyme's affinity for different tRNA substrates varies, with tRNAs containing U-starting anticodons typically showing 2-5 fold lower Km values than other tRNAs, reflecting the enzyme's biological specificity.

How does MiaA function impact D. vulgaris stress response?

While specific information about MiaA's role in D. vulgaris stress response is limited in the search results, we can draw insights from the organism's known stress adaptation mechanisms:

• Oxidative stress: D. vulgaris possesses genes like rubredoxin:oxygen oxidoreductase (roo) and hybrid cluster protein (hcp) that enhance survival under microaerobic conditions . MiaA-catalyzed tRNA modifications likely support efficient translation of stress response proteins during oxidative stress.

• Nitrite stress: D. vulgaris can reduce nitrite under certain conditions, with hybrid cluster proteins (Hcp) playing a critical role in alleviating nitrite stress by maintaining electron transport chain integrity . Proper MiaA function may be particularly important for translating these specific stress response proteins.

• Temperature adaptation: tRNA modifications often contribute to thermal stability of tRNA structure. MiaA-catalyzed modifications could help maintain translation efficiency during temperature fluctuations.

• Growth phase regulation: In many bacteria, tRNA modification levels change with growth phase, potentially allowing MiaA to act as a regulatory element connecting nutrient availability to translation efficiency.

The genomic island in D. vulgaris that contains stress-response genes like roo1 and hcp1 contributes significantly to the organism's fitness in microaerobic environments . This suggests that mechanisms supporting protein synthesis, including MiaA-catalyzed tRNA modification, are likely integrated with the organism's broader stress response systems.

What approaches can determine the MiaA-tRNA co-crystal structure?

Determining the co-crystal structure of D. vulgaris MiaA with its tRNA substrate requires:

• Protein-tRNA complex preparation:

  • Generate catalytically inactive MiaA (via site-directed mutagenesis)

  • Prepare homogeneous tRNA (either in vitro transcribed or purified from cells)

  • Form stable complex by incubating MiaA with tRNA at 1:1.2 molar ratio

  • Isolate the complex via size exclusion chromatography

• Crystallization screening:

  • Initial screening: Sparse matrix screens at 4°C and 18°C

  • Optimization: Vary pH (6.5-8.5), precipitant concentration, and additives

  • Microseeding to improve crystal quality

  • Use of nucleic acid-specific additives (e.g., spermine, cobalt hexammine)

• Data collection and processing:

  • Cryo-protection optimization to prevent ice formation

  • Collection at synchrotron radiation source

  • Processing with programs like XDS, DIALS, or HKL-2000

• Structure solution:

  • Molecular replacement using existing MiaA structures as search models

  • Careful model building of the tRNA portion

  • Refinement with programs that handle protein-nucleic acid complexes

• Validation and analysis:

  • Validate protein-RNA interactions

  • Identify key recognition elements

  • Compare with structures of homologous enzymes

The central channel structure observed in P. aeruginosa DMATase suggests that capturing the MiaA-tRNA complex might reveal how the enzyme positions the tRNA for modification while allowing DMAPP to enter from the opposite side.

How can inhibitors of D. vulgaris MiaA be identified and characterized?

A systematic approach to identify and characterize D. vulgaris MiaA inhibitors includes:

• Inhibitor discovery strategies:

  • Structure-based virtual screening targeting the DMAPP binding site

  • Fragment-based screening using thermal shift assays

  • High-throughput screening of compound libraries using activity assays

  • Substrate analog design (DMAPP mimetics)

• Primary screening assays:

  • Pyrophosphate release assay (coupled colorimetric reaction)

  • Fluorescence-based activity assays

  • Thermal shift assays for binding identification

• Secondary validation:

  • IC50 and Ki determination

  • Mode of inhibition studies (competitive, noncompetitive, uncompetitive)

  • Structure-activity relationship analysis

• Structural characterization:

  • Co-crystallization with inhibitors

  • NMR studies to identify binding sites

  • Molecular dynamics simulations to understand binding dynamics

• Selectivity profiling:

  • Testing against homologous enzymes from other organisms

  • Cellular activity assessment

Understanding the ordered binding mechanism of substrates (tRNA first, DMAPP second) is crucial when designing inhibition studies, as it affects experimental design and interpretation of kinetic data for different inhibitor types.

How can recombinant D. vulgaris MiaA be expressed in E. coli?

A detailed protocol for E. coli expression of recombinant D. vulgaris MiaA:

• Construct design:

  • Codon-optimize the D. vulgaris miaA gene for E. coli expression

  • Include N-terminal His6-tag with TEV protease cleavage site

  • Clone into pET28a vector between NdeI and XhoI sites

• Transformation and expression:

  • Transform into E. coli BL21(DE3)

  • Grow in LB medium with 50 μg/mL kanamycin at 37°C to OD600 = 0.6-0.8

  • Cool culture to 18°C before induction

  • Induce with 0.5 mM IPTG

  • Continue growth at 18°C for 18 hours

• Cell harvest and lysis:

  • Harvest cells by centrifugation (5,000 × g, 10 min, 4°C)

  • Resuspend in lysis buffer: 50 mM Tris-HCl pH 8.0, 300 mM NaCl, 10% glycerol, 5 mM β-mercaptoethanol, 1 mM PMSF

  • Lyse by sonication (6 × 30 sec, 50% amplitude, with 30 sec cooling between pulses)

  • Clarify lysate by centrifugation (30,000 × g, 30 min, 4°C)

• Quality control checkpoints:

  • Verify expression by SDS-PAGE analysis of pre- and post-induction samples

  • Assess solubility by comparing whole cell, supernatant, and pellet fractions

  • Confirm identity by Western blot using anti-His antibody

This protocol typically yields 15-20 mg of soluble recombinant MiaA per liter of culture, with expression levels verified by SDS-PAGE analysis showing a prominent band at approximately 35-40 kDa.

What analytical methods can verify D. vulgaris MiaA-catalyzed tRNA modification?

Multiple analytical approaches can confirm and characterize MiaA-catalyzed tRNA modifications:

• HPLC analysis:

  • Enzyme digest of modified tRNA using nuclease P1 and alkaline phosphatase

  • Reverse-phase HPLC separation of nucleosides

  • Detection by UV absorbance (typically at 254 nm)

  • Comparison with synthetic i6A standard

  • Typical retention time for i6A: 15-20 minutes on C18 column with methanol gradient

• Mass spectrometry approaches:

  • LC-MS/MS analysis of digested tRNA

  • Expected mass shift: +68 Da for i6A compared to adenosine

  • Characteristic fragmentation pattern of i6A

  • MALDI-TOF analysis of RNase digestion products

• Thin-layer chromatography (TLC):

  • 32P-labeled tRNA digestion products

  • Two-dimensional TLC using specific solvent systems

  • Autoradiographic detection

• Antibody-based detection:

  • Northern blot using antibodies specific for i6A

  • Dot blot assays for high-throughput screening

• NMR spectroscopy:

  • For detailed structural characterization of the modification

  • Requires purification of larger quantities of modified tRNA

The choice of method depends on the specific research question, with mass spectrometry offering the highest sensitivity and specificity for confirming the presence of the modification.

How can tRNA substrates be prepared for in vitro studies with D. vulgaris MiaA?

Preparing suitable tRNA substrates for in vitro MiaA assays involves:

• In vitro transcription method:

  • Clone tRNA gene into pUC19 under T7 promoter

  • Linearize plasmid with BstNI for precise 3' end

  • Transcribe using T7 RNA polymerase with reaction conditions:

    • 40 mM Tris-HCl pH 8.0, 22 mM MgCl2

    • 1 mM spermidine, 5 mM DTT

    • 4 mM each NTP

    • DNA template at 20 μg/mL

    • T7 RNA polymerase at 50 μg/mL

  • Incubate 3-4 hours at 37°C

  • Purify by denaturing PAGE (12%)

• Native tRNA purification:

  • Isolate total tRNA from D. vulgaris or E. coli

  • Enrich specific tRNAs using affinity chromatography with biotinylated oligonucleotides

  • Verify purity by denaturing PAGE and Northern blotting

• Quality control tests:

  • Aminoacylation assay to verify functional competence

  • Thermal melting analysis to confirm proper folding

  • 5' and 3' end homogeneity analysis

• Special considerations:

  • For accurate kinetic studies, ensure >95% purity

  • Proper refolding by heating to 80°C followed by slow cooling in the presence of Mg2+

  • Confirm absence of modification at A37 position by HPLC analysis of nucleosides

In vitro transcribed tRNAs lack post-transcriptional modifications, making them ideal substrates for studying MiaA activity, while native purified tRNAs provide insights into the enzyme's behavior with naturally occurring tRNA structures.

What are the common troubleshooting strategies for D. vulgaris MiaA activity assays?

When encountering issues with D. vulgaris MiaA activity assays, consider these troubleshooting approaches:

• Low or no enzymatic activity:

  • Verify enzyme integrity by SDS-PAGE and thermal shift assay

  • Ensure tRNA substrate is properly folded (heat and slow cool with Mg2+)

  • Check for inhibitory contaminants in the reaction buffer

  • Confirm DMAPP quality by mass spectrometry

  • Test different Mg2+ concentrations (1-10 mM range)

  • Add fresh reducing agent (DTT or β-mercaptoethanol)

  • Verify pH optimum (typically pH 7.5-8.0)

• Inconsistent results between replicates:

  • Standardize tRNA refolding procedure

  • Use master mixes to minimize pipetting errors

  • Control reaction temperature precisely

  • Verify linearity of detection method

  • Check for nuclease contamination

• High background in radioactive assays:

  • Increase washing stringency of filter-binding steps

  • Include higher concentrations of non-specific competitors

  • Perform additional purification of [14C]-DMAPP

  • Include appropriate negative controls (heat-inactivated enzyme)

• Problems with substrate quality:

  • tRNA: verify intact 3' CCA end by acid urea gel electrophoresis

  • DMAPP: store in small aliquots at -80°C to prevent degradation

  • Exclude metal contaminants with chelating resin treatment

• Activity loss during storage:

  • Add stabilizers (10% glycerol, 0.1 mg/mL BSA)

  • Avoid freeze-thaw cycles by preparing single-use aliquots

  • Test stability at different storage temperatures

Maintaining the ordered binding mechanism (tRNA first, DMAPP second) is crucial for activity, so ensure assay conditions support this sequential substrate binding process.

How can molecular dynamics simulations provide insights into D. vulgaris MiaA function?

Molecular dynamics (MD) simulations offer valuable insights into D. vulgaris MiaA structure-function relationships:

• System preparation:

  • Build homology model based on P. aeruginosa DMATase crystal structure

  • Include explicit solvent and physiological ion concentrations

  • Perform energy minimization and equilibration

• Simulation approaches:

  • Standard MD: 100-500 ns simulations to sample conformational space

  • Steered MD: Model substrate entry through the central channel

  • Free energy calculations: Determine binding energetics of substrates

  • Essential dynamics: Identify correlated motions relevant to catalysis

• Key phenomena to investigate:

  • Conformational changes upon tRNA binding

  • DMAPP access to the active site through the channel

  • Water and ion movement in the active site

  • Dynamics of the ordered binding mechanism

  • Effects of mutations on protein dynamics

• Analysis methods:

  • Root mean square deviation/fluctuation (RMSD/RMSF) analysis

  • Principal component analysis of domain movements

  • Hydrogen bond network analysis

  • Solvent accessible surface area calculations

  • Binding free energy calculations

• Validation approaches:

  • Compare simulation predictions with experimental mutagenesis results

  • Verify dynamic behavior with hydrogen-deuterium exchange experiments

  • Correlate computational findings with kinetic data

Simulations can particularly help understand how the central channel architecture facilitates the ordered entry of substrates from opposite sides and coordinates the catalytic reaction at the center of the enzyme.

How has the MiaA enzyme evolved across bacterial species?

Evolutionary analysis of MiaA across bacterial species reveals:

• Phylogenetic distribution:

  • Present in nearly all bacteria, indicating fundamental importance

  • Higher sequence conservation in the catalytic domain than in peripheral regions

  • Clustering that generally follows taxonomic classifications

• Structural conservation:

  • The central channel architecture seen in P. aeruginosa DMATase is likely conserved across bacterial MiaA enzymes

  • Conservation of the ordered binding mechanism (tRNA first, DMAPP second)

  • Homology with small soluble kinases involved in nucleotide biosynthesis, suggesting evolutionary origin

• Adaptation to different tRNA pools:

  • Subtle variations in tRNA-binding regions correlate with differences in organismal tRNA content

  • Thermophilic bacteria possess MiaA variants with additional stabilizing features

• Functional divergence:

  • Some bacterial MiaA enzymes show broader substrate specificity

  • Variations in kinetic parameters reflect adaptation to different cellular environments

  • Regulatory mechanisms controlling MiaA expression differ across bacterial phyla

The relationship to kinases involved in nucleotide biosynthesis provides insight into how this enzyme family evolved from basic metabolic enzymes to specialized tRNA modification catalysts while maintaining core structural features like the central substrate channel.

What is the relationship between MiaA function and other tRNA modification pathways?

MiaA functions within a broader network of tRNA modification enzymes:

• Sequential modification pathways:

  • MiaA catalyzes the first step (i6A formation) in a pathway that can continue with:

    • MiaB: thiomethylation to form ms2i6A

    • MiaE: hydroxylation to form io6A

  • These modifications work together to fine-tune tRNA function

• Cooperative effects with other modifications:

  • Synergistic effects with modifications at other positions (e.g., position 34)

  • Structural stabilization allowing other modifications to occur

  • Combined contributions to translational efficiency and accuracy

• Regulatory interplay:

  • Coordinated regulation of modification enzymes under stress conditions

  • Potential competition for the same tRNA substrates

  • Sequential dependency where certain modifications require prior modifications

• Evolutionary co-variation:

  • Correlated presence/absence patterns across bacterial species

  • Co-evolution of interacting modification systems

  • Adaptation to specific translational needs

In D. vulgaris, proper tRNA modification likely supports stress response mechanisms similar to how hybrid cluster proteins (Hcp) maintain electron transport chain integrity under nitrite stress , with each modification system contributing to the organism's ability to adapt to changing environmental conditions.

How do tRNA modifications by MiaA affect translation efficiency and fidelity?

MiaA-catalyzed tRNA modifications influence translation through several mechanisms:

• Anticodon-codon interactions:

  • The i6A modification at position 37 enhances base stacking

  • Stabilizes the first base pair of the codon-anticodon interaction

  • Particularly important for U-starting codons (UNN)

  • Reduces frameshifting errors

• Decoding kinetics:

  • Accelerates the rate of correct codon recognition

  • Slows incorrect codon recognition

  • Enhances discrimination between cognate and near-cognate codons

• Quantitative effects on translation:

  • Increases translation efficiency by 2-4 fold for specific codons

  • Most pronounced effect on rare codons

  • Critical for efficient translation of specific stress response proteins

• Global proteomic effects:

  • Alters translation of specific subsets of mRNAs

  • Particularly affects proteins with high content of UNN codons

  • Can function as a regulatory mechanism under stress conditions

These effects are particularly relevant in specialized organisms like D. vulgaris, where precise control of protein expression under changing environmental conditions is essential for survival, similar to how other systems like rubredoxin:oxygen oxidoreductase enhance survival under microaerobic conditions .

What computational tools can predict MiaA substrate specificity?

Several computational approaches can predict and analyze MiaA substrate specificity:

• Sequence-based methods:

  • Position-specific scoring matrices (PSSMs) for tRNA features

  • Support vector machines trained on known substrates

  • Neural networks incorporating sequence and structural features

  • Typical accuracy: 75-85% for predicting MiaA substrates

• Structure-based approaches:

  • Molecular docking of tRNA models to MiaA structures

  • Monte Carlo simulations of enzyme-substrate complexes

  • Free energy calculations for binding affinity prediction

  • Analysis of complementary electrostatic surfaces

• Integrated prediction pipelines:

  • tRNAscan-SE for tRNA gene identification

  • MODOMICS database integration for modification mapping

  • Custom scripts combining sequence and structural features

  • Web servers for automated prediction (e.g., HAMR, tRNAmod)

• Key features for predictions:

  • Presence of adenosine at position 37

  • Anticodon sequence context

  • tRNA tertiary structure stability

  • Anticodon stem-loop flexibility

Understanding the central channel architecture of MiaA has improved computational models by helping researchers incorporate the spatial constraints of substrate entry and positioning in their predictions.

How does D. vulgaris MiaA compare to eukaryotic tRNA isopentenyltransferases?

D. vulgaris MiaA and eukaryotic tRNA isopentenyltransferases (IPTases) show important similarities and differences:

• Structural comparison:

  • Both contain a central catalytic domain

  • Bacterial MiaA (like P. aeruginosa DMATase) features a central channel with substrates entering from opposite sides

  • Eukaryotic IPTases have additional regulatory domains

  • Both share evolutionary relationship with nucleotide kinases

• Substrate specificity:

  • Bacterial MiaA: primarily modifies tRNAs with adenosine at position 37

  • Eukaryotic IPTases: more selective, often modifying only a subset of tRNAs with A37

  • Both preferentially modify tRNAs reading codons that begin with U

• Catalytic mechanism:

  • Both use DMAPP as isoprenoid donor

  • Shared ordered binding mechanism (tRNA first)

  • Conserved catalytic residues despite sequence divergence

  • Similar metal ion requirements

• Biological context:

  • Bacterial MiaA: generally constitutive expression

  • Eukaryotic IPTases: often subject to complex regulation

  • Differential subcellular localization in eukaryotes

  • Expanded roles in eukaryotic cellular signaling

The fundamental similarity in core structure and mechanism suggests that bacterial MiaA and eukaryotic IPTases evolved from a common ancestor, while differences in peripheral domains and regulation reflect adaptation to the more complex eukaryotic cellular environment.