Recombinant Azorhizobium caulinodans tRNA pseudouridine synthase A (truA)

What is truA and what role does it play in Azorhizobium caulinodans?

TruA (tRNA pseudouridine synthase A) is an enzyme that catalyzes the isomerization of specific uridines in cellular RNAs to pseudouridines (Ψ) . In A. caulinodans, this enzyme (EC 5.4.99.12) is also known as tRNA pseudouridine(38-40) synthase, tRNA pseudouridylate synthase I, or tRNA-uridine isomerase I . The full-length protein consists of 258 amino acids and is involved in modifying specific positions in tRNA molecules, which is crucial for their proper folding and function . This enzyme likely plays important roles during both free-living conditions and during symbiotic nitrogen fixation, as A. caulinodans forms nitrogen-fixing nodules on stems and roots of Sesbania rostrata, which is particularly important in lowland rice cultivation under flood conditions .

How is truA expression regulated in Azorhizobium caulinodans under different conditions?

While the specific regulation of truA in A. caulinodans under different conditions is not directly addressed in the available data, genome-wide transcriptional profiling has been performed on this organism under various conditions . A whole-genome microarray analysis compared gene expression in free-living cells grown in rich and minimal media versus bacteroids isolated from stem nodules . In such studies, genes involved in sulfur uptake and metabolism, acetone metabolism, and the biosynthesis of exopolysaccharide showed higher expression in bacteroids compared to free-living cells . Further research would be needed to specifically analyze truA expression patterns during the transition from free-living to symbiotic states.

What is the biochemical mechanism of pseudouridylation by truA?

Pseudouridine synthases like truA catalyze the isomerization of specific uridines to pseudouridines in RNA without requiring external energy sources or cofactors . The reaction involves breaking the glycosidic bond between the uracil base and ribose, rotating the base by 180 degrees, and reforming the bond, resulting in a carbon-carbon bond rather than the original nitrogen-carbon bond . This modification enhances base-stacking capabilities and provides additional hydrogen-bonding potential, contributing to RNA structural stability . Pseudouridine synthases may also function as RNA chaperones, helping RNAs achieve their proper three-dimensional structures .

How does the structure and function of truA compare to other tRNA modification enzymes?

Pseudouridine synthases are classified into different families (including TruA, TruB, TruC, TruD, and RsuA), all of which are descended from a common molecular ancestor despite having different substrate specificities . The 1.85 Å resolution structure of TruB, a related pseudouridine synthase, reveals that these enzymes recognize the three-dimensional structure of their RNA targets primarily through shape complementarity and access their substrate uridines by flipping out the nucleotide from the RNA structure .

TruA specifically modifies positions 38-40 in the anticodon stem-loop of tRNAs, whereas other family members target different positions. For example, TruB is responsible for the pseudouridine residue present in the T loops of virtually all tRNAs . Unlike some other RNA modification enzymes that require guide RNAs (such as box H/ACA snoRNPs with the Cbf5/dyskerin catalytic subunit), truA can directly recognize its substrate .

What approaches can be used to analyze truA activity in relation to nitrogen fixation symbiosis?

To investigate the role of truA in the nitrogen-fixing symbiotic relationship between A. caulinodans and S. rostrata, researchers could employ several approaches:

• Gene knockout studies: Creating truA deletion mutants to observe effects on symbiosis establishment and nitrogen fixation efficiency. This could be analyzed similar to the hydrogenase studies in search result , where specific gene deletions were created and their effects on symbiotic function were measured.

• Comparative transcriptomics: Analyzing truA expression during different stages of nodule formation and comparing this with other genes known to be important for symbiosis. Whole-genome microarray analysis has already been used to compare gene expression between free-living and symbiotic states of A. caulinodans .

• Protein localization studies: Using fluorescent protein fusions or immunolocalization to determine where truA is located within bacteroids in nodules.

• Structure-function analysis: Using site-directed mutagenesis to identify residues critical for truA function and then observing how these mutations affect symbiotic performance.

• Metabolomic analysis: Investigating whether changes in tRNA modification patterns correlate with metabolic shifts during symbiosis establishment.

How might truA influence bacterial adaptation to symbiotic environments?

TruA-mediated tRNA modifications could influence A. caulinodans adaptation to symbiotic environments in several ways:

• Translational efficiency: Pseudouridylation of tRNAs can enhance translation accuracy and efficiency, which may be crucial during the metabolic reprogramming that occurs when bacteria transition from free-living to symbiotic states .

• Stress response: Within plant nodules, bacteria encounter different environmental conditions, including potential oxidative stress and microaerobic conditions . tRNA modifications have been implicated in stress responses in many organisms and could help A. caulinodans adapt to these challenges.

• Host-specific protein synthesis: The expression of specific proteins required for nitrogen fixation and symbiosis may be optimized by particular tRNA modification patterns.

• Gene regulation: Changes in tRNA modification could potentially influence which codons are most efficiently translated, providing an additional layer of gene regulation during symbiosis.

What kinetic parameters characterize truA enzymatic activity?

While the specific kinetic parameters for A. caulinodans truA are not provided in the available data, enzymatic analysis would typically include:

• Substrate specificity: Determining which tRNA species and specific uridine positions are preferentially modified by truA.

• Kinetic constants: Measuring Km (substrate concentration at half-maximal velocity), kcat (turnover number), and kcat/Km (catalytic efficiency) under various conditions.

• Reaction conditions: Establishing optimal pH, temperature, and ionic requirements for enzymatic activity.

• Inhibition patterns: Identifying molecules that inhibit truA activity and characterizing the type of inhibition (competitive, noncompetitive, etc.).

Similar enzyme kinetic approaches have been used to characterize other enzymes, as shown in search result where mutational analysis was combined with activity measurements to understand catalytic mechanisms.

What structural features enable truA to recognize specific tRNA substrates?

Based on insights from related pseudouridine synthases, truA likely recognizes its tRNA substrates through a combination of factors :

• Shape complementarity: The enzyme probably recognizes the preformed three-dimensional structure of specific tRNA regions.

• Nucleotide flipping: As observed with TruB, truA likely accesses its target uridines by flipping them out of the RNA structure .

• Sequence recognition: Specific amino acid residues may interact with nucleotides surrounding the target uridine to ensure modification at the correct position.

• RNA chaperone activity: Beyond its catalytic function, truA may help stabilize or promote specific RNA conformations that are recognized by the enzyme .

Additional structural studies would be needed to precisely determine which amino acid residues in A. caulinodans truA are involved in substrate recognition and catalysis.

What are the optimal conditions for expressing and purifying recombinant A. caulinodans truA?

Based on the product information for recombinant A. caulinodans truA, the following guidelines should be considered :

• Expression system: The recombinant protein described in search result is produced using a baculovirus expression system, suggesting that insect cell expression may be optimal for maintaining proper folding and activity.

• Purification: The protein can be purified to >85% purity as determined by SDS-PAGE .

• Storage conditions: Store at -20°C, or for extended storage, conserve at -20°C or -80°C. Repeated freezing and thawing is not recommended, and working aliquots can be stored at 4°C for up to one week .

• Reconstitution protocol:

  • Briefly centrifuge the vial before opening to bring contents to the bottom

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

  • Add 5-50% glycerol (final concentration) and aliquot for long-term storage

  • The default final concentration of glycerol is 50%

• Shelf life: The shelf life of the liquid form is approximately 6 months at -20°C/-80°C, while the lyophilized form has a shelf life of 12 months at -20°C/-80°C .

What assays can be used to measure truA activity in vitro?

Several methods can be employed to measure the pseudouridylation activity of truA in vitro:

• Mass spectrometry-based assays: Similar to the approach mentioned in search result , mass spectrometry can be used to monitor enzymatic activities in real-time. This approach could detect the mass difference between uridine and pseudouridine within RNA substrates.

• Radioisotope incorporation: Using radiolabeled RNA substrates to track the conversion of uridine to pseudouridine, with detection by techniques such as thin-layer chromatography or high-performance liquid chromatography.

• Chemical derivatization: Pseudouridine can be specifically labeled with certain chemical reagents that don't react with uridine, allowing for selective detection of the modified nucleoside.

• NMR spectroscopy: Nuclear magnetic resonance can be used to directly observe the structural changes that occur during pseudouridylation.

• Fluorescence-based assays: Development of fluorescent probes that respond to pseudouridylation events could provide a high-throughput method for measuring truA activity.

These assays would typically be performed using purified recombinant truA and synthetic or natural tRNA substrates under controlled conditions.

How can site-directed mutagenesis be used to identify catalytic residues in truA?

Site-directed mutagenesis is a powerful approach for investigating enzyme catalytic mechanisms. For truA, this would involve:

• Identifying candidate catalytic residues: Based on sequence alignments with other pseudouridine synthases or structural predictions, amino acids potentially involved in catalysis or substrate binding can be identified.

• Creating specific mutations: Multiple types of mutations can be informative:

  • Alanine substitutions to remove the functional group

  • Conservative substitutions (e.g., Asp to Glu) to test the importance of specific chemical properties

  • Charge-reversal mutations to test electrostatic interactions

• Activity assays with mutant proteins: Comparing the activity of wild-type and mutant proteins can reveal which residues are essential for catalysis or substrate binding.

A similar approach was used in search result for another enzyme (UppP), where mutations like E21A, S26A, S27A, and R174A were created to disrupt the catalytic site and analyze their effects on enzyme function.

What RNA substrates are most suitable for in vitro studies of truA?

For in vitro studies of truA activity, several RNA substrate options could be considered:

• Full-length tRNAs: Natural tRNA molecules containing the target uridines at positions 38-40 would provide the most physiologically relevant substrates. These could be either purified from cells or produced by in vitro transcription.

• Synthetic tRNA fragments: Shorter RNA oligonucleotides containing the anticodon stem-loop region with the target uridines might be sufficient and easier to work with than full-length tRNAs.

• Structurally defined RNA constructs: Since pseudouridine synthases recognize the three-dimensional structure of their targets , designing RNA substrates with the proper structural features is crucial.

• Species-specific tRNAs: Using tRNAs from A. caulinodans itself would be most relevant for understanding the natural function of truA in this organism.

When selecting RNA substrates, it's important to consider that recognition by truA likely depends on both sequence elements and three-dimensional structure, as indicated by studies of related pseudouridine synthases .

How does A. caulinodans truA compare with tRNA modification enzymes in other nitrogen-fixing bacteria?

While the available data doesn't provide a direct comparison of truA across different nitrogen-fixing bacteria, such a comparative analysis would be valuable for understanding evolutionary adaptations in tRNA modification systems. A. caulinodans has some unique properties among rhizobia, including its ability to form nitrogen-fixing nodules on both stems and roots of its host legume and having the smallest genome and symbiosis island among sequenced rhizobia (5.4 Mb and 86.7 kb, respectively) .

A comprehensive comparative analysis would examine:

• Sequence conservation: Alignment of truA sequences from various nitrogen-fixing bacteria to identify conserved and divergent regions.

• Substrate specificity: Determining whether truA enzymes from different species modify the same tRNA positions.

• Expression patterns: Comparing truA expression during symbiosis across different bacterial-plant partnerships.

• Genomic context: Analyzing whether truA is located near other genes involved in symbiosis or tRNA modification in different species.

What techniques can be used to study truA function in vivo?

To study truA function within living A. caulinodans cells and during symbiosis, researchers could employ:

• Gene deletion and complementation: Creating truA knockout mutants and observing phenotypic effects, followed by complementation with the wild-type gene to confirm specificity.

• Conditional expression systems: Using inducible promoters to control truA expression at different stages of growth or symbiosis.

• RNA-seq analysis: Comparing transcriptome-wide changes in truA mutants versus wild-type bacteria.

• tRNA modification profiling: Using mass spectrometry to quantify pseudouridylation levels in various tRNAs under different conditions or in different genetic backgrounds.

• Fluorescent protein fusions: Creating truA-GFP fusions to track protein localization during different growth phases and symbiotic stages.

• Symbiosis phenotyping: Assessing nodulation efficiency, nitrogen fixation rates, and plant growth promotion by truA mutants compared to wild-type bacteria, similar to approaches used for hydrogenase studies in A. caulinodans .

How might truA contribute to adaptation during environmental stress?

TruA-mediated tRNA modifications could play important roles in bacterial adaptation to environmental stresses:

• Oxidative stress response: Within nodules, bacteria may encounter reactive oxygen species. Pseudouridylation could enhance translational accuracy under oxidative stress conditions.

• Temperature adaptation: A. caulinodans must adapt to temperature fluctuations in soil and plant environments, and tRNA modifications often contribute to temperature adaptability.

• Microaerobic adaptation: As A. caulinodans is described as a microaerophilic bacterium , truA might facilitate adaptation to low-oxygen environments encountered during symbiosis.

• Nutrient limitation: During transitions between free-living and symbiotic states, bacteria experience changes in nutrient availability. tRNA modifications could optimize translation efficiency under different nutrient conditions.

Experimental approaches to investigate these possibilities could include exposing wild-type and truA mutant strains to various stresses and comparing survival rates, growth characteristics, and global translation patterns.

What are the potential applications of recombinant truA in RNA biology research?

Recombinant truA has several potential applications in RNA biology research:

• RNA structure-function studies: As pseudouridylation affects RNA structure and stability, recombinant truA could be used to introduce specific modifications into RNA molecules for structural and functional studies.

• Synthetic biology applications: Engineered tRNA modification patterns could potentially enhance protein expression systems or expand the genetic code.

• RNA therapeutics: Understanding and potentially manipulating pseudouridylation could have applications in RNA-based therapeutics.

• Evolutionary studies: Comparing the substrate specificities of truA enzymes from different organisms could provide insights into the evolution of RNA modification systems.

• Biotechnology applications: If tRNA modifications enhance translation under stress conditions, engineered truA variants could potentially improve the performance of bacteria in biotechnological applications.

These applications would require detailed understanding of truA's catalytic mechanism, substrate specificity, and the functional consequences of pseudouridylation in different RNA contexts.