Recombinant Thermus thermophilus (Dimethylallyl)adenosine tRNA methylthiotransferase MiaB (miaB)

What is the fundamental role of tRNA modifications in Thermus thermophilus?

Thermus thermophilus employs multiple tRNA modifications to adapt protein synthesis to environmental temperature changes. Three distinct modifications (Gm18, m5s2U54, and m1A58) have been well-characterized and together increase the melting temperature of tRNA by approximately 10°C compared to unmodified transcripts . These modifications function as structural stabilizers that alter tRNA rigidity depending on environmental conditions. Interestingly, the level of these modifications varies with growth temperature - cells cultured at 80°C show higher modification levels than those cultured at 50°C, demonstrating an adaptive mechanism to optimize protein synthesis across temperature ranges .

What experimental systems are available to study Thermus thermophilus tRNA modifications?

Researchers studying Thermus thermophilus tRNA modifications have access to several established experimental systems, including:

• Genetic manipulation through transformation protocols using plasmids like pMKPnqosGFP with selection via antibiotics such as kanamycin (30 μg/mL)

• RNA sequencing methodologies specifically optimized for thermophiles

• Gene knockout strategies, as demonstrated with the HB27Δago strain

• Comparative analysis between wild-type and genetically modified strains under various growth conditions

• Commercial availability of recombinant proteins like MiaB for in vitro studies

How do temperature-dependent tRNA modifications affect translational efficiency in Thermus thermophilus?

Research has demonstrated that temperature-dependent tRNA modifications directly impact translational efficiency in T. thermophilus. tRNA isolated from cells grown at 80°C efficiently synthesizes poly(U) at high temperatures (above 65°C), while tRNA from cells grown at 50°C functions optimally at lower temperatures . This remarkable adaptation involves strategic modification of tRNA structure through enzymes like MiaB, allowing the translation machinery to maintain functionality across a wide temperature range (50-83°C). To investigate this phenomenon, researchers should design experiments comparing translational efficiency using tRNAs isolated from cells grown at different temperatures, examining both rate and accuracy metrics.

What is the relationship between MiaB activity and other tRNA modification enzymes in stress response?

The interrelationship between MiaB and other tRNA modification enzymes represents a sophisticated stress response system. Research suggests that rather than simple transcriptional or translational regulation, the coordination between these enzymes may involve complex RNA recognition mechanisms . For example, studies have shown that disruption of genes for modifications like m7G46 and ψ55 (trmB and truB) can affect the levels of other modifications such as Gm18, m5s2U54, and m1A58 . Experiments designed to investigate these relationships should include:

• Sequential enzyme knockout studies measuring downstream effects on modification patterns

• Time-course analyses of modification enzyme activities following temperature shifts

• Structural studies examining enzyme-substrate recognition under various stress conditions

• Systems biology approaches to map the entire modification network

How can structural analysis of thermostable MiaB inform protein engineering strategies?

The structural features that enable Thermus thermophilus MiaB to function at high temperatures make it an excellent model for protein engineering. Understanding the molecular basis of its thermostability could inform strategies to enhance the stability of other proteins. Research approaches should include:

• Comparative structural analysis between thermophilic and mesophilic MiaB homologs

• Identification of stabilizing interactions (hydrogen bonds, salt bridges, hydrophobic interactions)

• Assessment of structural rigidity versus flexibility at catalytically important regions

• Directed evolution experiments to further enhance thermostability or alter substrate specificity

What experimental approaches can reveal the precise catalytic mechanism of Thermus thermophilus MiaB?

Elucidating the catalytic mechanism of T. thermophilus MiaB requires sophisticated experimental approaches due to the complex nature of methylthiotransferase reactions. Researchers should consider:

• Pre-steady-state kinetic measurements to identify rate-limiting steps

• Spectroscopic techniques (EPR, Mössbauer) to characterize iron-sulfur cluster intermediates

• Isotope labeling studies to track sulfur and methyl group transfers

• X-ray crystallography and cryo-EM studies of enzyme-substrate complexes at different reaction stages

• Computational simulation of radical-based reaction mechanisms at elevated temperatures

What are the optimal conditions for expressing and purifying recombinant Thermus thermophilus MiaB?

Successful expression and purification of T. thermophilus MiaB requires careful consideration of its thermophilic origin and likely iron-sulfur cluster content. Based on protocols used for similar thermophilic enzymes, researchers should consider:

• Expression systems:

  • E. coli BL21(DE3) with co-expression of iron-sulfur cluster assembly proteins

  • T. thermophilus HB27 as a homologous expression host (growth at 65°C)

  • Anaerobic expression conditions to protect iron-sulfur clusters

• Purification strategy:

  • Heat treatment (65-70°C) to remove mesophilic host proteins

  • Anaerobic purification to maintain iron-sulfur cluster integrity

  • Buffer optimization containing reducing agents and stabilizing additives

• Quality control methods:

  • UV-visible spectroscopy to confirm iron-sulfur cluster presence

  • Activity assays at elevated temperatures (65-80°C)

  • Mass spectrometry to confirm protein integrity and cofactor binding

How can RNA-seq be optimized for studying the effects of MiaB on Thermus thermophilus transcriptome?

RNA sequencing can be a powerful tool for investigating how MiaB affects the T. thermophilus transcriptome. Based on established methodologies:

• Sample preparation:

  • Cultivation of wild-type and miaB-knockout strains under identical conditions

  • RNA extraction using specialized protocols for thermophiles (e.g., mirVana RNA isolation kit)

  • Quality control with bioanalyzer to ensure RNA integrity

• Sequencing considerations:

  • Prepare biological triplicates for statistical robustness

  • Map reads against the T. thermophilus genome (HB27 chromosome and pTT27 mega-plasmid)

  • Use differential expression analysis tools (e.g., trinity package with RSEM)

• Data analysis approaches:

  • Identify genes with altered expression patterns in the absence of MiaB

  • Categorize affected transcripts by function and codon usage

  • Correlate findings with other tRNA modification systems

What approaches can be used to generate and validate miaB knockout strains in Thermus thermophilus?

Creating miaB knockout strains requires specialized techniques suitable for thermophiles:

• Knockout generation:

  • Natural competence-based transformation (T. thermophilus is naturally competent)

  • Homologous recombination with selection cassettes

  • Growth medium supplementation with kanamycin (30 μg/mL) for selection

• Validation methods:

  • PCR verification of genomic modifications

  • RNA-seq to confirm altered expression patterns

  • Mass spectrometry of tRNA to confirm absence of specific modifications

  • Phenotypic assays comparing growth rates at different temperatures

How can researchers characterize the temperature-dependent activity profile of MiaB?

Understanding the temperature-dependent activity of MiaB is essential for comprehending its biological function:

• In vitro activity assays:

  • Purified enzyme assays across temperature range (30-90°C)

  • Substrate (tRNA) modification analysis by HPLC or mass spectrometry

  • Determination of kinetic parameters (kcat, KM) at different temperatures

• Stability assessments:

  • Differential scanning calorimetry to determine melting temperature

  • Circular dichroism to monitor secondary structure changes with temperature

  • Activity half-life measurements at different temperatures

• Structural dynamics investigations:

  • Hydrogen-deuterium exchange mass spectrometry at various temperatures

  • Molecular dynamics simulations predicting temperature-dependent conformational changes

  • NMR studies of protein flexibility at different temperatures

Temperature-Dependent tRNA Modification Levels in Thermus thermophilus

Comparative Analysis of tRNA Functionality at Different Temperatures

Key Enzymes in the Thermus thermophilus tRNA Modification Network