Recombinant Bradyrhizobium japonicum tRNA pseudouridine synthase B (truB)

What is the molecular function of Bradyrhizobium japonicum truB and how does it compare to other bacterial pseudouridine synthases?

Bradyrhizobium japonicum tRNA pseudouridine synthase B (truB) is an enzyme responsible for introducing pseudouridine at position 55 of tRNAs during the early stages of tRNA maturation. This modification is critical for enhancing base-to-base stacking and stabilizing tRNA structure . Similar to its Escherichia coli counterpart, B. japonicum truB introduces pseudouridine at the conserved position 55 in the TΨC loop of tRNAs, which is one of the first RNA modifications discovered in tRNA and ribosomes .

Unlike other pseudouridine synthases such as PUS10, which modifies positions 54 and 55 of tRNA in archaea, B. japonicum truB demonstrates high specificity for position 55 . Research indicates that truB can function beyond its enzymatic activity as an RNA chaperone, helping tRNAs achieve their proper folding independent of pseudouridylation activity .

What are the critical residues involved in enzymatic activity and RNA binding of Bradyrhizobium japonicum truB?

Studies on conserved residues between bacterial truB homologs have identified several critical amino acids essential for both enzymatic activity and RNA binding capability:

These residues were identified based on high conservation between E. coli TruB and B. japonicum TruB . Mutation experiments have demonstrated that substitutions at these positions generate distinct phenotypes: mutations at D48 and D90 create catalytically inactive enzymes that can still bind RNA substrates, while mutations at K64 eliminate RNA binding capability while maintaining protein structure .

How can the pseudouridylation activity of recombinant Bradyrhizobium japonicum truB be measured in vitro?

The pseudouridylation activity of recombinant B. japonicum truB can be measured through several complementary approaches:

• Direct enzymatic assay: Measure the conversion of uridine to pseudouridine using radiolabeled tRNA substrates followed by nuclease digestion and thin-layer chromatography to separate and quantify nucleotides .

• CMC-primer extension assay: Treat RNA with N-cyclohexyl-N'-(2-morpholinoethyl)carbodiimide metho-p-toluenesulfonate (CMC), which specifically attaches to pseudouridine residues, followed by primer extension. The reverse transcriptase stops at the CMC-modified pseudouridine, creating a distinct band on sequencing gels that indicates pseudouridine position .

• EMSA (Electrophoretic Mobility Shift Assay): Assess RNA binding capability by incubating recombinant truB protein with tRNA substrates and analyzing the resulting complexes on native gels to visualize RNA-protein interactions .

The CMC-primer extension method is particularly sensitive for detecting pseudouridylation in low-abundance RNA samples and provides precise positional information about pseudouridine modifications.

What is the optimal expression system for producing recombinant Bradyrhizobium japonicum truB protein?

For optimal expression of recombinant B. japonicum truB, a bacterial expression system using E. coli BL21(DE3) with the following parameters has proven effective:

The slow growth at reduced temperature is particularly important for obtaining properly folded, enzymatically active truB protein. The enzyme requires proper folding of its characteristic trefoil knot structure, which is critical for its catalytic function .

How does Bradyrhizobium japonicum truB contribute to bacterial survival and symbiotic relationships?

B. japonicum truB plays multiple crucial roles in bacterial physiology and symbiotic relationships:

• tRNA stabilization: Pseudouridylation at position 55 enhances tRNA structural stability, which is particularly important under stress conditions encountered during host colonization .

• Translation fidelity: Properly modified tRNAs reduce translational frameshifting and ensure accurate protein synthesis, which is essential for the expression of symbiosis-related genes .

• Adaptation to environmental stress: TruB activity may be regulated in response to environmental changes, including those encountered during the transition from soil to plant host environments .

The importance of truB for B. japonicum's symbiotic capability is suggested by the bacterium's remarkable ability to survive saprophytically in soil for extended periods (over 20 years) while maintaining nodulation competence . This long-term survival may depend on proper tRNA modification and maintenance of translation fidelity under varying environmental conditions.

What are the differences between Bradyrhizobium japonicum truB and the eukaryotic pseudouridine synthases?

B. japonicum truB differs from eukaryotic pseudouridine synthases in several key aspects:

Unlike eukaryotic enzymes, bacterial truB enzymes use a unique trefoil knot structure for AdoMet binding and catalysis. This knot consists of three β-strands at the central β-sheet, creating a structural fold that enables the enzyme to bind its substrate in a distinctive manner .

What experimental approaches can be used to investigate truB's role in Bradyrhizobium japonicum physiology?

Several experimental approaches can effectively investigate truB's physiological role:

• Gene knockout studies: Generate truB deletion mutants and assess their growth, stress response, and symbiotic capabilities with host plants like soybeans .

• Complementation assays: Reintroduce wild-type or mutant truB genes into knockout strains to verify phenotype restoration and identify critical functional domains .

• RNA-Seq analysis: Compare transcriptomes of wild-type and truB mutant strains to identify genes affected by disruption of tRNA modification .

• Ribosome profiling: Assess translation efficiency and accuracy in truB mutants to determine effects on protein synthesis .

• Plant inoculation experiments: Test the ability of truB mutants to nodulate and fix nitrogen in symbiosis with legume hosts under controlled conditions .

Such approaches have successfully identified the functional roles of related proteins in Bradyrhizobium. For example, studies with different B. japonicum strains showed that mutations affecting symbiotic capabilities could be traced to specific genetic elements .

How can the RNA substrates of Bradyrhizobium japonicum truB be identified and characterized?

Comprehensive identification and characterization of RNA substrates can be achieved through:

• HITS-CLIP (High-Throughput Sequencing Crosslinking Immunoprecipitation): This technique allows genome-wide identification of RNAs that directly interact with truB in vivo. UV crosslinking covalently links the protein to its RNA targets, followed by immunoprecipitation and high-throughput sequencing .

• In vitro pseudouridylation assays: Test various RNA substrates (tRNAs, rRNAs, mRNAs) with purified recombinant truB to determine modification specificity .

• Comparative RNA-Seq with pseudouridine-specific chemical labeling: Treat RNA with CMC, which binds specifically to pseudouridine, followed by reverse transcription and sequencing to identify pseudouridylation sites transcriptome-wide .

Studies with other bacterial truB proteins have shown that while tRNAs are the primary substrate, some mRNAs may also contain pseudouridine modifications. HITS-CLIP analysis for TruB1 revealed physical association with both tRNAs and a series of mRNAs, though functional significance of these interactions varies .

What is the structure of the Bradyrhizobium japonicum truB protein and how does it relate to function?

The B. japonicum truB protein contains a distinctive structural feature known as a "trefoil knot" that is essential for its function:

• Trefoil knot structure: This topological feature consists of three β-strands (β3, β4, and β5) at the central β-sheet. The knot starts with β3, followed by a loop that turns at the back of β3 and emerges into β4. Another loop follows β4 and turns into β5, which makes a circular insertion into the knot by crossing over β3 .

• AdoMet binding: The trefoil knot binds S-adenosyl methionine (AdoMet) in an unusual bent shape, constraining the adenosine and methionine moieties to face each other, which is critical for methyl transfer .

• Cross-chain communication: The trefoil knot is also vital for intermolecular signaling between the two monomers of truB, coordinating activity for efficient catalysis .

Mutations within the trefoil knot region can render the enzyme temperature-sensitive and catalytically compromised. For example, a mutation at position S88 (at the beginning of the central β3 strand leading into the trefoil knot) significantly affects enzyme function .

How does the activity of truB differ between various Bradyrhizobium japonicum strains?

TruB activity can vary significantly between different B. japonicum strains:

These variations may contribute to the different ecological adaptations observed in various Bradyrhizobium strains. For instance, the dominance patterns of B. japonicum strains vary with geographical distribution, with USDA 123 being prevalent in northern regions of the United States, while B. elkanii strains dominate in middle to southern regions .

The truB enzyme may play a role in these adaptation patterns by ensuring proper protein synthesis through tRNA modification, particularly under the stress conditions encountered during soil survival and plant colonization .

What are the methods for analyzing the impact of truB mutations on Bradyrhizobium japonicum growth and symbiotic capabilities?

To analyze the impact of truB mutations on B. japonicum:

• Growth curve analysis: Compare doubling times of wild-type and mutant strains under various conditions using least squares fitting exponential calculations for accurate determination of growth rates .

• Nodulation assays: Inoculate host plants like Lotus strigosus or soybeans with wild-type and mutant strains, then quantify nodule number, size, and distribution after 8 weeks of growth .

• Plant growth effects: Measure both absolute and relative plant growth parameters (shoot/root biomass) compared to uninoculated controls to assess symbiotic efficiency .

• Nitrogen fixation assessment: Use acetylene reduction assays to quantify nitrogenase activity as a measure of functional nitrogen fixation in nodules .

• Competitive fitness assays: Perform co-inoculation experiments with wild-type and mutant strains to assess competitive ability for nodule formation under field-relevant conditions .

These approaches have been successfully used to characterize other mutations in B. japonicum, revealing how specific genetic changes affect symbiotic performance .

How does the Mg²⁺ dependence of bacterial truB affect its function in Bradyrhizobium japonicum?

The Mg²⁺ dependence of bacterial truB is a distinctive feature with significant functional implications:

This Mg²⁺ requirement makes bacterial truB distinct from eukaryotic pseudouridine synthases and potentially more susceptible to environmental changes in metal ion availability, which may be significant during the transition between free-living and symbiotic lifestyles .

What techniques can be used to study the interaction between recombinant Bradyrhizobium japonicum truB and its RNA substrates?

Several specialized techniques are effective for studying truB-RNA interactions:

• EMSA (Electrophoretic Mobility Shift Assay): This technique can assess RNA binding by incubating recombinant truB with RNA substrates and analyzing the resulting complexes on native gels. It provides information about binding affinity and can be quantified to determine dissociation constants .

• Filter binding assays: Using radiolabeled RNA substrates to measure the retention of RNA-protein complexes on nitrocellulose filters, allowing quantitative analysis of binding parameters .

• Surface Plasmon Resonance (SPR): This label-free technology can determine association and dissociation rates of truB-RNA interactions in real-time .

• HITS-CLIP: For in vivo analysis, this technique allows identification of RNA interaction sites through UV crosslinking, immunoprecipitation, and sequencing .

Studies using these approaches with related proteins have revealed that bacterial truB primarily recognizes the tRNA through interactions with the phosphodiester backbone rather than through direct base recognition, a mechanism known as "indirect readout" .

How can recombinant truB be used to study the evolutionary relationships between different Bradyrhizobium species?

Recombinant truB provides valuable insights into Bradyrhizobium evolution through:

• Comparative sequence analysis: Analyzing truB gene sequences across Bradyrhizobium species reveals evolutionary relationships and potential adaptation to different environments .

• Functional conservation testing: Expressing recombinant truB from different species and testing their activity on standardized substrates can reveal functional divergence .

• Complementation studies: Introducing truB genes from different Bradyrhizobium species into truB-deficient strains to test functional conservation across species .

Such studies align with research showing distinct distributions of Bradyrhizobium species across different geographical regions. For instance, B. japonicum Bj123 dominates in the northern United States, while B. elkanii is more prevalent in middle to southern regions . Analyzing truB functional differences may provide insights into these distribution patterns and the selective pressures driving Bradyrhizobium evolution.

What are the dual functions of truB in bacterial physiology beyond tRNA modification?

Research indicates that truB has important dual functions beyond its primary role in tRNA modification:

• RNA chaperone activity: TruB functions as an RNA chaperone independent of its pseudouridylation activity, helping tRNAs and potentially other RNAs achieve proper folding. This chaperone function appears to be mechanistically distinct from its enzymatic activity .

• Potential regulatory roles: Similar to findings with the related protein TruB1, which was found to regulate let-7 miRNA maturation, bacterial truB may influence regulatory RNA processing beyond tRNA modification .

• Stress response: TruB may participate in bacterial stress responses through its RNA modification and chaperone activities, particularly under conditions that challenge RNA stability, such as temperature fluctuations encountered during host infection .