Recombinant Corynebacterium glutamicum tRNA (Ile)-lysidine synthase (tilS)

What is tRNA (Ile)-lysidine synthase (TilS) in Corynebacterium glutamicum and what role does it play in bacterial translation?

TilS (systematic name: L-lysine:(tRNAIle2)-cytidine34 ligase (AMP-forming)) is an essential enzyme in C. glutamicum that catalyzes the post-transcriptional modification of the wobble position of tRNAIle2. Specifically, TilS modifies cytidine34 in the anticodon by adding lysine to form lysidine . This critical modification converts the codon recognition specificity from AUG (methionine) to AUA (isoleucine) and changes the amino acid specificity for synthetase activation from methionine to isoleucine .

The catalytic reaction can be represented as:
[tRNAIle2]-cytidine34 + L-lysine + ATP → [tRNAIle2]-lysidine34 + AMP + diphosphate + H2O

This modification is essential for bacterial viability as it ensures proper translation of the isoleucine codon AUA. Without this modification, ribosomes would be unable to translate past AUA codons, disrupting protein synthesis .

How does the structure of C. glutamicum TilS compare to TilS enzymes from other bacterial species?

While the search results don't provide specific structural information about C. glutamicum TilS, several high-resolution protein structures of bacterial TilS are available . The functional domains of TilS are generally conserved across bacterial species.

Methodological approach for structural comparison:

• Obtain the amino acid sequence of C. glutamicum TilS

• Perform multiple sequence alignment with TilS proteins from other bacterial species (e.g., E. coli, B. subtilis)

• Use structural prediction software to model C. glutamicum TilS

• Compare the predicted structure with available crystal structures

• Focus on the active site residues and binding domains for tRNAIle2, ATP, and lysine

Research findings indicate that TilS homologs are nearly universally present in eubacteria, including human pathogens . The conservation of this enzyme across bacterial species, coupled with its absence in mammals, makes it a potential target for broad-spectrum antibacterial agents .

What are the essential substrates required for TilS activity and how do they interact?

TilS requires three essential substrates for its catalytic activity:

• tRNAIle2 with a CAU anticodon - The specific target for modification

• ATP - Provides energy for the reaction

• L-lysine - The amino acid that gets attached to the cytidine34

The enzyme interaction with these substrates follows a sequential mechanism. In kinetic studies of TilS from A. aeolicus, the Km values were determined to be:

• ATP: Variable concentrations from 40–640 μM were tested

• tRNAIle2 transcript: Variable concentrations from 2.5–156.8 μM were tested

• L-lysine: Variable concentrations from 0.25–4 mM were tested, with WT enzyme having a Km value of 629 μM

Methodological approach:
To study substrate interactions, researchers typically employ a combination of:

• Steady-state kinetics to determine Km and kcat values

• Isothermal titration calorimetry to measure binding affinities

• Site-directed mutagenesis to identify key residues involved in substrate binding

• X-ray crystallography of enzyme-substrate complexes

How can mutations in the tilS gene affect C. glutamicum metabolism and adaptation to different growth conditions?

Mutations in the tilS gene can have significant effects on C. glutamicum metabolism and adaptation. Research has shown that tilS mutations can influence:

• Adaptation to overflow metabolism: Mutations that impair tRNA modification by TilS have been observed to provide fitness advantages under conditions favoring rapid, redox-imbalanced growth .

• Lag phase reduction: Both tilS mutations and mutations in tRNAIle2 decreased lag phase duration by approximately 3.5 hours, conferring a competitive advantage over wild-type strains .

• Growth enhancement under specific conditions: The fitness advantages of tilS mutations were specific to rapid growth on galactose using oxidative overflow metabolism that generates redox imbalance, not resources favoring more balanced metabolism .

Methodological approach for studying tilS mutations:

• Generate targeted mutations in tilS using CRISPR-Cas9 or traditional homologous recombination

• Conduct growth experiments under various conditions (different carbon sources, oxygen levels)

• Measure growth parameters (lag phase, growth rate, final biomass)

• Analyze global gene expression using RNA-seq

• Assess the level of lysidine-modified tRNAIle2 using mass spectrometry

Research has shown that compromised TilS activity appears to improve growth under specific metabolic conditions, suggesting that TilS may have secondary functions beyond ensuring translational accuracy .

What experimental approaches can be used to assess the kinetic parameters of recombinant C. glutamicum TilS?

Several experimental approaches can be used to determine the kinetic parameters of recombinant C. glutamicum TilS:

• Radioactive assay using [U-14C] L-lysine:

  • Reaction buffer: 100 mM Tris·HCl (pH 7.8), 10 mM MgCl₂, 10 mM KCl, 10 mM DTT

  • Fixed concentrations of two substrates while varying the third

  • Spotting reaction aliquots on filter paper soaked with TCA

  • Washing filters with TCA and ethanol to remove unreacted substrate

  • Measuring radioactivity by scintillation counter

• Aminoacylation assay:

  • Using purified IleRS to assess if lysidine-modified tRNAIle2 can be charged with isoleucine

  • This confirms the functional consequence of the TilS modification

• Gel electrophoresis assay:

  • Analyzing mobility shifts of tRNAIle2 before and after TilS modification

  • This approach can also be used to test alternative substrates or inhibitors

For accurate kinetic analysis, the following experimental design considerations are important:

• Ensure enzyme concentration is in the linear range of activity

• Control temperature carefully (typically 30-60°C depending on the source organism)

• Include proper controls (no enzyme, no substrate)

• Use Lineweaver-Burk plots or non-linear regression to calculate Km and kcat values

When analyzing C. glutamicum TilS specifically, researchers should consider the unique membrane and metabolic characteristics of this organism that might affect enzyme activity in vivo .

How does lysidine modification by TilS affect the aminoacylation of tRNAIle2 in C. glutamicum?

The lysidine modification catalyzed by TilS is critical for proper aminoacylation of tRNAIle2. Research has confirmed that:

• Lysidine modification is both necessary and sufficient to convert tRNAIle2 into a substrate for isoleucyl-tRNA synthetase (IleRS) .

• Without lysidine modification, tRNAIle2 with a CAU anticodon would be recognized by methionyl-tRNA synthetase instead of IleRS, leading to misincorporation of methionine at isoleucine codons .

• The modification changes both codon recognition (from AUG to AUA) and amino acid specificity (from methionine to isoleucine) .

Methodological approach for studying aminoacylation:

• Prepare in vitro transcribed tRNAIle2

• Perform TilS reaction on a portion of the tRNA

• Conduct aminoacylation assays with both modified and unmodified tRNA using purified IleRS and MetRS

• Analyze charging efficiency using acid gel electrophoresis or filter binding assays with radiolabeled amino acids

Experimental evidence from studies with purified recombinant E. coli TilS and in vitro transcribed tRNA substrate has confirmed that lysidine modification is both necessary and sufficient for IleRS recognition .

What are the specific factors affecting the efficiency of recombinant C. glutamicum TilS expression systems?

Several factors influence the efficiency of recombinant C. glutamicum TilS expression:

• Expression host:

  • E. coli is commonly used for heterologous expression

  • C. glutamicum itself can be used for homologous expression

  • Each system has different advantages for codon usage, post-translational modifications, and protein folding

• Expression vector elements:

  • Promoter strength (constitutive vs. inducible)

  • Signal peptides for potential secretion

  • Affinity tags for purification (His-tag, FLAG-tag)

• Growth conditions:

  • Temperature (typically lower for better folding)

  • Induction parameters (concentration, timing)

  • Media composition (rich vs. minimal)

• Protein solubility:

  • Addition of solubility-enhancing tags (MBP, SUMO)

  • Co-expression with chaperones

  • Optimization of lysis buffers

Methodological considerations from research:
Based on the expression of other proteins in C. glutamicum, researchers have observed that:

• The IPTG-inducible Ptrc promoter can be effective for controlled expression

• Signal peptides like CgR0949 can direct transport via the Tat-dependent pathway

• C-terminal affinity tags (6xHis) can facilitate purification and detection

• The efficiency of heterologous protein expression in C. glutamicum can be affected by signal peptide choice, promoter strength, and the nature of the protein

How can a lysidine formation assay be set up to measure C. glutamicum TilS activity?

A robust lysidine formation assay can be established using the following protocol:

Reagents and materials:

• Purified recombinant C. glutamicum TilS

• In vitro transcribed tRNAIle2

• ATP

• [U-14C] L-lysine (for radioactive assay)

• Buffer components: Tris·HCl, MgCl₂, KCl, DTT

Reaction setup:

• Prepare reaction buffer: 100 mM Tris·HCl (pH 7.8), 10 mM MgCl₂, 10 mM KCl, 10 mM DTT

• Add substrates: 2 mM ATP, 10-40 μM tRNAIle2 transcript, 250-4000 μM [U-14C] L-lysine

• Initiate reaction by adding purified TilS (0.125-4 μM, depending on activity)

• Incubate at optimal temperature (30-60°C)

• Remove aliquots at defined time points (e.g., 2, 5, and 10 min)

Detection methods:

• Radioactive filter binding assay:

  • Spot reaction aliquots on filter paper pre-soaked with TCA

  • Wash filters with ice-cold 5% TCA (three times) and ethanol (twice)

  • Measure radioactivity by scintillation counting

• Alternative methods:

  • Mass spectrometry to directly detect lysidine formation

  • Aminoacylation assay with IleRS to test functional activity

  • Gel mobility shift assay

Data analysis:

• Calculate initial velocities from time-course data

• Use Lineweaver-Burk plots or non-linear regression to determine kinetic parameters

• Compare activities across different experimental conditions

This assay has been successfully used with TilS from other bacterial species and can be adapted for C. glutamicum TilS .

What are the best methods for purifying recombinant C. glutamicum TilS while maintaining enzymatic activity?

Purifying recombinant C. glutamicum TilS with preserved enzymatic activity requires careful consideration of expression systems and purification strategies:

Expression system optimization:

• Construct design:

  • Add N-terminal or C-terminal His-tag or FLAG-tag for affinity purification

  • Consider using fusion proteins (MBP, SUMO) to enhance solubility

  • Include a precision protease cleavage site for tag removal

• Expression conditions:

  • Test various induction parameters (temperature, inducer concentration, duration)

  • Lower temperatures (16-25°C) often improve proper folding

  • Rich media supplemented with cofactors may enhance expression

Purification protocol:

• Cell lysis:

  • Buffer composition: 50 mM Tris-HCl (pH 7.5-8.0), 300 mM NaCl, 10% glycerol, 1 mM DTT

  • Include protease inhibitors to prevent degradation

  • Gentle lysis methods (sonication with cooling periods or enzymatic lysis)

• Affinity chromatography:

  • Immobilized metal affinity chromatography (IMAC) for His-tagged proteins

  • Anti-FLAG affinity chromatography for FLAG-tagged proteins

  • Gradual elution to separate different binding populations

• Additional purification steps:

  • Ion exchange chromatography to separate based on charge

  • Size exclusion chromatography to remove aggregates and ensure homogeneity

  • Heparin affinity chromatography, which can be effective for nucleic acid-binding proteins

• Activity preservation:

  • Include stabilizing agents (glycerol, reducing agents) in all buffers

  • Determine protein stability at different pH values and salt concentrations

  • Consider adding small amounts of substrate (e.g., ATP) to stabilize the enzyme

  • Avoid multiple freeze-thaw cycles; store as aliquots at -80°C

Based on successful purification of other recombinant proteins from C. glutamicum, a FLAG-tag system has been effectively used for purification and detection, as demonstrated in the purification of IleRS from B. subtilis .

How can in vitro transcribed tRNAIle2 be prepared as a substrate for C. glutamicum TilS assays?

Preparing high-quality in vitro transcribed tRNAIle2 for TilS assays involves several critical steps:

Template preparation:

• Obtain the C. glutamicum tRNAIle2 sequence and design overlapping oligonucleotides that cover the entire tRNA

• Include a T7 RNA polymerase promoter at the 5' end

• Add a restriction site (e.g., BstNI) at the 3' end for linearization

• Clone the sequence into a suitable vector (e.g., pLDR24)

• Verify the sequence by DNA sequencing

In vitro transcription:

• Linearize the plasmid with the appropriate restriction enzyme

• Set up transcription reaction:

  • 40 mM Tris-HCl (pH 8.0)

  • 22 mM MgCl₂

  • 5 mM DTT

  • 1 mM spermidine

  • 4 mM each NTP

  • DNA template

  • T7 RNA polymerase

  • RNase inhibitor

  • Pyrophosphatase to prevent inhibition by pyrophosphate

• Incubate at 37°C for 3-4 hours

Purification of tRNA transcript:

• Extract with phenol/chloroform and precipitate with isopropanol

• Purify by denaturing (urea) 10% PAGE

• Further purify by HPLC using a Resource Q column

• Verify purity and integrity by gel electrophoresis

Functional verification:

• Test a small amount of the purified tRNA in a TilS assay

• Check for proper folding using structure probing techniques

• Verify that the tRNA can be modified by TilS and subsequently aminoacylated by IleRS

This methodology has been successfully used to prepare tRNAIle2 transcripts for studying TilS from A. aeolicus and E. coli .

What approaches can be used to study the impact of TilS mutations on C. glutamicum physiology and protein translation?

Studying the impact of TilS mutations on C. glutamicum physiology and protein translation requires a multi-faceted approach:

Generation of TilS mutations:

• Site-directed mutagenesis for targeted mutations

• Random mutagenesis (e.g., error-prone PCR or ARTP mutagenesis) for broad screening

• CRISPR-Cas9 genome editing for chromosomal modifications

• Plasmid-based expression of mutant TilS variants

Phenotypic characterization:

• Growth analysis:

  • Measure growth parameters (lag phase, growth rate, final biomass)

  • Test various carbon sources and stress conditions

  • Competitive growth assays with wild-type strains

• Metabolic profiling:

  • Analyze amino acid production/consumption (especially lysine and isoleucine)

  • Measure central carbon metabolites

  • Examine byproduct formation

Molecular analysis of translation:

• Quantification of lysidine levels:

  • Mass spectrometry-based detection of lysidine in tRNA pools

  • Northern blot analysis with specific probes for modified/unmodified tRNAIle2

• Translation accuracy assessment:

  • Reporter systems using AUA codons in critical positions

  • Proteomic analysis to detect mistranslation events

  • Ribosome profiling to identify translation pausing at AUA codons

• Enzyme activity assays:

  • Purify mutant TilS proteins and measure kinetic parameters

  • Determine the impact on substrate binding and catalysis

Global impact analysis:

• Transcriptomic analysis (RNA-seq) to identify compensatory responses

• Proteomic analysis to detect changes in protein expression

• Ribosome profiling to examine global translation dynamics

Research findings have shown that TilS mutations can provide fitness advantages under specific growth conditions, suggesting secondary functions beyond translational accuracy . For example, mutations in TilS have been observed to decrease lag phase by approximately 3.5 hours and create competitive advantages specifically during rapid, redox-imbalanced growth .

What are common challenges in expressing recombinant C. glutamicum TilS and how can they be addressed?

Researchers commonly encounter several challenges when expressing recombinant C. glutamicum TilS. Here are the major issues and their solutions:

Challenge 1: Low expression levels
Solutions:

• Optimize codon usage for the expression host

• Try different promoters (e.g., IPTG-inducible Ptrc promoter has been successful for recombinant protein expression in C. glutamicum)

• Adjust induction conditions (temperature, inducer concentration, induction time)

• Test different expression hosts (E. coli BL21(DE3), C. glutamicum)

• Use stronger ribosome binding sites to enhance translation initiation

Challenge 2: Protein insolubility
Solutions:

• Lower induction temperature (16-25°C)

• Co-express with molecular chaperones

• Add solubility-enhancing tags (MBP, SUMO, GST)

• Include stabilizing agents in lysis buffer (glycerol, reduced glutathione)

• Express as a fusion protein with a highly soluble partner

Challenge 3: Protein instability/degradation
Solutions:

• Include protease inhibitors during purification

• Use protease-deficient host strains

• Identify and modify protease recognition sites in the protein sequence

• Optimize buffer conditions (pH, salt concentration, additives)

• Store with glycerol or other stabilizing agents

Challenge 4: Low enzymatic activity
Solutions:

• Ensure proper protein folding by optimizing expression conditions

• Add cofactors or substrate analogs during purification to stabilize active conformation

• Test activity immediately after purification to minimize storage-related activity loss

• Verify that the affinity tag doesn't interfere with activity (consider tag removal)

• Check for post-translational modifications that might be required for activity

Challenge 5: Contamination with host tRNAs
Solutions:

• Include high-salt washes during purification

• Use nuclease treatment followed by heat inactivation

• Employ additional purification steps (ion exchange, size exclusion)

• Verify purity by testing activity with and without adding tRNA substrate

Research indicates that while C. glutamicum has potential as a host for heterologous protein production, low protein yields and high levels of native protein secretion can be significant challenges . When expressing TilS specifically, ensuring that the enzyme retains its ability to bind tRNAIle2 and catalyze lysidine formation is crucial for downstream applications.

How can researchers troubleshoot inconsistent results in TilS activity assays?

When troubleshooting inconsistent results in TilS activity assays, researchers should systematically evaluate each component of the experimental system:

Enzyme quality issues:

• Verify enzyme purity by SDS-PAGE

• Check for batch-to-batch variation in enzyme preparations

• Determine enzyme stability under storage conditions

• Test activity of fresh versus stored enzyme preparations

• Verify that the enzyme concentration is in the linear range of the assay

Substrate quality issues:

• Confirm tRNA folding by native gel electrophoresis

• Verify ATP quality (degradation can occur during freeze-thaw cycles)

• Ensure radiolabeled lysine has not degraded (check specific activity)

• Prepare fresh substrate solutions for critical experiments

Assay condition variables:

• Maintain consistent temperature throughout the reaction

• Control pH carefully (buffer capacity and preparation)

• Verify metal ion concentrations (especially Mg²⁺)

• Ensure consistent timing for all reaction steps

• Test different reaction vessels (plastic versus glass binding effects)

Detection method troubleshooting:
For radioactive filter binding assays:

• Ensure consistent filter washing procedures

• Check background binding of free [14C]-lysine to filters

• Verify scintillation counter performance with standards

• Use multiple technical replicates

Systematic approach to troubleshooting:

• Test positive and negative controls in each experiment

• Vary one parameter at a time

• Perform time-course experiments to identify reaction phases

• Use alternative detection methods to cross-validate results

• Develop a standard curve with known amounts of enzyme

Based on published protocols, the lysidine formation assay should be performed at a defined temperature (e.g., 60°C) in a reaction buffer containing 100 mM Tris·HCl (pH 7.8), 10 mM MgCl₂, 10 mM KCl, and 10 mM DTT . When calculating kinetic parameters, it's important to keep two substrate concentrations fixed while varying the third to obtain reliable Lineweaver-Burk plots .

What strategies can address potential contamination issues when purifying recombinant C. glutamicum TilS?

Contamination during TilS purification can compromise both enzyme purity and activity. Here are comprehensive strategies to address various contamination issues:

Microbial contamination:

• Work under sterile conditions whenever possible

• Use sterile-filtered buffers and solutions

• Add bacteriostatic agents (e.g., sodium azide at 0.02%) to buffers

• Clean all equipment thoroughly before use

• Include sterility testing as part of quality control

Nucleic acid contamination:

• Treat protein samples with nucleases (e.g., Benzonase)

• Include high salt washes (500 mM - 1 M NaCl) during affinity purification

• Use polyethyleneimine precipitation to remove nucleic acids

• Employ anion exchange chromatography to separate protein from nucleic acids

• Verify absence of nucleic acids by measuring A260/A280 ratio (should approach 0.6 for pure protein)

Host protein contamination:

• Design expression constructs with highly specific affinity tags

• Use stringent washing conditions during affinity purification

• Employ multiple orthogonal purification techniques (e.g., affinity followed by ion exchange and size exclusion)

• Consider subtractive approaches to remove specific contaminants

• Verify purity by SDS-PAGE and mass spectrometry

Endotoxin contamination (for downstream cellular applications):

• Use endotoxin-free reagents and plasticware

• Include Triton X-114 phase separation steps

• Use polymyxin B-based endotoxin removal columns

• Test final preparation with LAL (Limulus Amebocyte Lysate) assay

• Include endotoxin removal specific resins in purification workflow

Cross-contamination between samples:

• Use dedicated columns and equipment for each preparation

• Employ thorough cleaning and sanitization between runs

• Include blank runs to verify absence of carryover

• Test for activity from potential contaminants

• Use unique affinity tags for different protein constructs

Metal ion contamination:

• Use high-quality reagents and ultrapure water

• Include EDTA in initial purification steps, followed by specific metal reconstitution if needed

• Test metal-free preparations for residual activity

• Use plastic labware to avoid metal leaching from glass

• Include a specific chelation step if metal contamination is suspected

When working with recombinant C. glutamicum proteins, it's important to consider that the bacterium produces high levels of native secreted proteins . Therefore, especially when expressing and purifying TilS from C. glutamicum itself rather than heterologous hosts, additional purification steps may be necessary to separate the target enzyme from these abundant native proteins.

How can researchers modify TilS to improve its catalytic efficiency or alter its substrate specificity?

Researchers can employ various strategies to engineer TilS for enhanced catalytic efficiency or altered substrate specificity:

Rational design approaches:

• Structure-guided mutagenesis:

  • Target residues in the active site that interact with substrates

  • Modify residues involved in rate-limiting steps

  • Enhance protein stability through disulfide bridges or salt bridges

  • Based on available TilS structures, focus on the ATP-binding pocket, lysine-binding site, and tRNA recognition elements

• Substrate binding optimization:

  • Alter the tRNA recognition domain to improve binding kinetics

  • Modify the lysine binding pocket to accommodate lysine analogs

  • Engineer the ATP binding site for more efficient catalysis

Directed evolution strategies:

• Random mutagenesis:

  • Error-prone PCR to generate libraries of TilS variants

  • DNA shuffling between TilS enzymes from different bacterial species

  • Create focused libraries targeting specific domains

• Selection/screening methods:

  • Develop high-throughput assays for lysidine formation

  • Link TilS activity to cell survival through complementation assays

  • Use FACS-based screening with fluorescent reporters

Computational design:

• Molecular dynamics simulations to identify dynamic bottlenecks

• In silico screening of mutations predicted to enhance catalysis

• Energy landscape analysis to identify stabilizing mutations

Domain swapping and fusion approaches:

• Create chimeric enzymes with domains from TilS of thermophilic organisms for increased stability

• Fuse TilS with RNA-binding domains for enhanced tRNA recognition

• Engineer protein scaffolds to create proximity-based enhancement of multi-step reactions

Research findings on substrate specificity indicate that TilS can incorporate various lysine analogs, including simple alkyl amines, suggesting flexibility in the lysine-binding pocket that could be exploited for engineering efforts . Additionally, studies of TilS mutations that evolved naturally in long-term evolution experiments with E. coli provide insights into functionally important residues that could be targets for engineering .

What are the latest methodologies for investigating the interaction between TilS and tRNAIle2 in C. glutamicum?

Recent advancements have expanded the toolkit for studying TilS-tRNAIle2 interactions in C. glutamicum:

Biophysical methodologies:

• Surface Plasmon Resonance (SPR):

  • Quantitative measurement of binding kinetics (kon, koff)

  • Determination of binding affinity (KD)

  • Analysis of the effects of mutations on binding

• Microscale Thermophoresis (MST):

  • Label-free detection of binding in solution

  • Low sample consumption

  • Compatible with complex buffer conditions

• Isothermal Titration Calorimetry (ITC):

  • Direct measurement of binding thermodynamics

  • Determination of stoichiometry, enthalpy, and entropy

  • No labeling required

Structural methodologies:

• Cryo-electron microscopy (cryo-EM):

  • Visualization of TilS-tRNA complexes in near-native states

  • Resolution of dynamic intermediates during catalysis

  • No crystallization required

• NMR spectroscopy:

  • Analysis of protein-RNA dynamics in solution

  • Identification of residues involved in binding

  • Characterization of conformational changes upon binding

• X-ray crystallography:

  • High-resolution structures of TilS-tRNA complexes

  • Identification of specific atomic interactions

  • Template for structure-based drug design

Chemical biology approaches:

• UV crosslinking combined with mass spectrometry:

  • Identification of direct contact points between TilS and tRNA

  • Analysis of transient interactions

  • No need for protein or RNA modification

• SHAPE (Selective 2'-hydroxyl acylation analyzed by primer extension):

  • Probing of tRNA structure before and after TilS binding

  • Identification of structural changes in tRNA upon binding

  • Mapping of protected regions

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

  • Analysis of protein dynamics upon tRNA binding

  • Identification of regions undergoing conformational changes

  • Characterization of allosteric effects

Cellular methodologies:

• CRISPR-Cas9 base editing:

  • Precise modification of tilS gene or tRNAIle2 in vivo

  • Analysis of phenotypic consequences

  • Study of structure-function relationships in cellular context

• Fluorescence microscopy with labeled TilS and tRNA:

  • Visualization of intracellular localization

  • Analysis of co-localization patterns

  • Study of dynamics using techniques like FRAP

Experimental evidence from lysidine formation assays with tRNAIle2 mutants has demonstrated the importance of specific tRNA structural elements for TilS recognition . These methodologies can provide deeper insights into the molecular basis of this enzyme-substrate interaction in C. glutamicum.

How can researchers study the role of TilS in translational fidelity across the C. glutamicum proteome?

Investigating the impact of TilS on translational fidelity across the C. glutamicum proteome requires sophisticated approaches that integrate genomics, proteomics, and computational analyses:

Ribosome profiling approaches:

• Ribo-seq analysis:

  • Compare wild-type and TilS-depleted/mutated strains

  • Analyze ribosome occupancy at AUA codons

  • Identify translational pausing sites

  • Quantify the efficiency of AUA translation

• Selective ribosome profiling:

  • Use antibiotics that capture specific translational states

  • Identify sites of miscoding or ribosome stalling

  • Compare profiles between normal and TilS-compromised conditions

Proteomics-based methods:

• Mass spectrometry for mistranslation detection:

  • Targeted analysis of isoleucine to methionine substitutions

  • SILAC labeling to quantify changes in the proteome

  • Analysis of post-translational modifications that might compensate for mistranslation

• Protein stability and aggregation:

  • Pulse-chase experiments to measure protein turnover rates

  • Analysis of insoluble protein fractions

  • Chaperone interaction profiling

Reporter systems:

• Fluorescent protein-based reporters:

  • Design reporters with critical AUA codons in functional positions

  • Dual-fluorescent protein systems to calculate mistranslation rates

  • Site-specific incorporation of unnatural amino acids at AUA positions

• Enzyme activity-based reporters:

  • Engineer enzymes where activity depends on correct isoleucine incorporation

  • High-throughput assays for enzyme function

Bioinformatic approaches:

• Codon usage analysis:

  • Examine the distribution of AUA codons across the genome

  • Identify genes with high AUA content

  • Correlate AUA usage with gene essentiality

• Comparative genomics:

  • Analyze conservation of tilS and tRNAIle2 across bacterial species

  • Identify compensatory mechanisms in species with different decoding strategies

Experimental design for studying TilS impact:

• Generate conditional TilS depletion strains:

  • Place tilS under control of inducible promoters

  • Create partial loss-of-function mutants

  • Use CRISPRi for targeted knockdown

• Analyze cellular responses:

  • Transcriptomic analysis to identify stress responses

  • Measure activation of quality control mechanisms

  • Examine growth phenotypes under different conditions