Recombinant Chromobacterium violaceum tRNA threonylcarbamoyladenosine biosynthesis protein RimN (rimN)

Basic Research Questions

• What is Chromobacterium violaceum tRNA threonylcarbamoyladenosine biosynthesis protein RimN?

RimN (also known as TsaC or YrdC) is a critical enzyme involved in the biosynthesis of threonylcarbamoyladenosine (t6A), a universal tRNA modification found at position 37 of nearly all tRNAs that decode ANN codons. In C. violaceum, this protein functions as an ATPase that binds to A37-containing tRNA and participates in the threonyl-carbamoylation process. The protein is part of a conserved family of enzymes present across all three domains of life, indicating its evolutionary significance in protein translation mechanisms .

• How does the t6A modification affect tRNA function in bacteria?

The t6A modification plays a crucial role in maintaining translational fidelity in bacteria. Specifically:

• It serves as a strong positive determinant for aminoacylation of tRNA by bacterial-type isoleucyl-tRNA synthetases

• It likely functions as a determinant for the essential enzyme tRNA Ile-lysidine synthetase (TilS)

• It helps maintain the accuracy of ANN codon recognition and prevents frameshifting during translation

• It stabilizes codon-anticodon interactions at the ribosomal A-site

The absence of t6A can lead to significant proteotoxic stress and widespread translational defects, particularly affecting proteins that contain ANN codons in critical positions .

• What is the structural organization of RimN/TsaC in C. violaceum?

RimN from C. violaceum shares structural similarities with other bacterial TsaC proteins. Based on structural data from homologous proteins:

The protein contains conserved motifs for nucleotide binding and has a characteristic fold that enables it to recognize specific features of the tRNA substrate .

Advanced Research Questions

• What experimental approaches are most effective for studying RimN function in C. violaceum?

Several complementary approaches can be used to study RimN function:

• Genetic approaches: Creating knockout mutants using transposon mutagenesis or CRISPR-Cas9 systems to assess the essentiality and phenotypic effects of RimN deletion

• Biochemical approaches: In vitro reconstitution of the t6A synthesis pathway using purified components

• Structural approaches: X-ray crystallography or cryo-EM to determine protein-tRNA interactions

• RNA analysis techniques: LC-MS/MS analysis of tRNA modifications to quantify t6A levels

• Transcriptomic analysis: RNA-seq to evaluate global effects of RimN deletion on gene expression

The combination of these approaches provides a comprehensive understanding of RimN function. For instance, reconstitution experiments have demonstrated that recombinant TsaC (RimN), along with TsaD, TsaB, and TsaE proteins, ATP, threonine, and bicarbonate, are collectively necessary and sufficient for t6A formation in tRNA substrates .

• How does RimN interact with other proteins in the t6A biosynthesis pathway?

RimN (TsaC) participates in a complex protein interaction network:

The interactions are hierarchical and species-specific, with the TsaC-TsaD interaction serving as the core of the complex. Notably, only YeaZ-YgjD pairs from closely related organisms form complexes in vitro, suggesting evolutionary constraints on these interactions .

• What is the mechanistic basis for the essentiality of RimN in bacteria but not in eukaryotes?

The differential essentiality of RimN between prokaryotes and eukaryotes stems from several factors:

• Aminoacyl-tRNA synthetase specificity: t6A is a strong positive determinant for aminoacylation by bacterial-type isoleucyl-tRNA synthetases but not for eukaryotic-type enzymes

• Decoding mechanisms: Bacterial ribosomes may have a stronger dependence on t6A for accurate decoding of ANN codons

• Alternative pathways: Eukaryotes may possess compensatory mechanisms that are absent in most bacteria

• TilS dependency: In bacteria, t6A may serve as a determinant for tRNA Ile-lysidine synthase (TilS), which is essential for AUA codon decoding

Experimental evidence shows that while t6A synthesis genes are essential in most prokaryotes, they are dispensable in certain bacterial species like Deinococcus radiodurans, Thermus thermophilus, Synechocystis PCC6803, and Streptococcus mutans .

• How can researchers effectively design experiments to study the role of RimN in C. violaceum pathogenicity?

To investigate the relationship between RimN and C. violaceum pathogenicity, researchers should design experiments with the following considerations:

Remember that C. violaceum is an environmental bacterium that can be occasionally pathogenic to humans and animals. Research has shown that C. violaceum infections are associated with soil and water exposure in tropical regions and can cause serious clinical infections with a mortality rate of approximately 7.1% .

Methodological Questions

• What are the optimal conditions for expressing and purifying recombinant RimN from C. violaceum?

Based on published protocols for homologous proteins, the following conditions are recommended:

For optimal activity, ensure the removal of contaminating nucleic acids during purification, as they can affect subsequent functional assays .

• What functional assays can be used to assess RimN activity in vitro?

Several complementary assays can be used to evaluate RimN enzymatic activity:

• ATP hydrolysis assay: Monitor AMP production using HPLC or coupled enzymatic assays

• tRNA binding assay: Use electrophoretic mobility shift assay (EMSA) or fluorescence anisotropy to measure binding affinity

• t6A formation assay: Direct measurement of t6A formation using:

  • LC-MS/MS analysis of modified tRNA

  • Radioisotope labeling with [14C]-threonine

  • Immunodetection with t6A-specific antibodies

• Reconstitution assays: Complete in vitro t6A formation using purified components including RimN, TsaD, TsaB, TsaE, ATP, threonine, and bicarbonate

For accurate activity measurement, use both A37-containing tRNA (natural substrate) and control tRNAs that do not naturally contain t6A, such as tRNA^Gln from Methanothermaobacter thermautotrophicus .

• How can site-directed mutagenesis be used to study RimN function?

Site-directed mutagenesis is a powerful approach for understanding structure-function relationships in RimN:

• Target selection: Choose residues based on:

  • Sequence conservation analysis across species

  • Structural information from crystal structures

  • Predicted functional domains (ATP binding, tRNA binding)

• Experimental approach:

  • Create single point mutations using standard PCR-based methods

  • Express and purify mutant proteins using the same protocol as wild-type

  • Compare enzymatic activities using functional assays

  • Conduct structural analysis to confirm the effect on protein folding

• Key residues to target:

  • ATP-binding pocket residues

  • Residues predicted to interact with tRNA

  • Interface residues that interact with other t6A synthesis proteins

This approach has been successfully applied to homologous proteins like MnmM, where mutations in tRNA-interacting residues such as K11E, D34A, N101D/A, Y104A, Y141F, and H144A resulted in significant reductions in enzymatic activity .

• How does the quorum sensing system in C. violaceum relate to RimN function?

While direct evidence linking the quorum sensing (QS) system to RimN function is limited, there are potential regulatory connections:

The CviI/R quorum sensing system in C. violaceum regulates numerous processes including violacein production, protease activity, and chitinase production. t6A modification and tRNA processing may be regulated in response to population density, potentially through:

• Transcriptional regulation: QS may influence the expression of genes involved in tRNA modification, including RimN

• Post-translational regulation: Population density signals might affect protein activity or complex formation

• Metabolic integration: QS-regulated metabolic shifts may indirectly affect the availability of substrates for t6A synthesis

Research has shown that in C. violaceum, the CviI/R system plays a significant role in regulating gene expression at high cell density, and this regulation is further modulated by proteins like VioS and the AirR system . Understanding how tRNA modification enzymes like RimN fit into this regulatory network represents an important research direction.

Integration with Broader Research Contexts

• How does understanding RimN function contribute to antimicrobial drug development?

RimN and the t6A synthesis pathway represent promising antimicrobial targets for several reasons:

• Selective targeting: The essential nature of t6A in most bacteria but not in eukaryotes offers selectivity for antibacterial agents

• Broad-spectrum potential: The conservation of RimN across bacterial species suggests broad-spectrum activity

• Novel mechanism: Targeting tRNA modification represents a mechanism distinct from existing antibiotics

• Reduced resistance potential: The essential and conserved nature of RimN may reduce the development of resistance

Notably, genes involved in t6A synthesis, including tsaC (RimN) and tsaD, had been identified as antibacterial targets even before their functions in t6A synthesis were known. Structural and functional studies of RimN provide crucial information for structure-based drug design approaches targeting this essential bacterial pathway .

• What technological advances have enabled recent progress in understanding tRNA modifications like t6A?

Several technological developments have accelerated research in tRNA modifications:

These advances have collectively enabled the elucidation of the complete t6A biosynthesis pathway and a better understanding of its role in bacterial physiology. For instance, comparative genomic-driven analysis allowed for the identification of the genes responsible for t6A synthesis, including RimN/TsaC .