Recombinant Desulfovibrio vulgaris Ribosomal RNA large subunit methyltransferase E (rlmE)

What is the primary function of RlmE in ribosomal RNA modification?

RlmE is responsible for the synthesis of Um2552 in the ribosomal RNA, a critical modification located adjacent to G2553, which is an essential base that anchors the 3' CCA terminus of the A-site tRNA in the peptidyl-transferase center (PTC). This methylation plays a crucial role in proper ribosome assembly and function, particularly in the large ribosomal subunit (LSU) . In bacterial systems like Escherichia coli, deletion of rlmE results in a notable decrease in growth rate (2–4-fold slower compared to wild-type cells), which suggests its important role in cellular function . While most research has focused on E. coli RlmE, the Desulfovibrio vulgaris homolog likely performs similar functions in rRNA modification based on sequence and structural conservation.

How do knockout studies inform our understanding of RlmE function?

Knockout studies have provided significant insights into the functional importance of RlmE. Unlike many other rRNA modification enzymes that can be individually knocked out with minimal growth effects, ΔrlmE strains exhibit noticeable growth defects and ribosome assembly issues. The severe assembly phenotype of ΔrlmE strains can be partially restored by overexpressing small GTPases (such as Obg and EngA), indicating that RlmE has functions in ribosome LSU assembly that extend beyond its methyltransferase activity . This suggests a dual role for RlmE: its catalytic function in rRNA modification and a structural role in ribosome assembly.

What expression systems are recommended for recombinant D. vulgaris RlmE production?

For recombinant expression of D. vulgaris RlmE, an E. coli-based expression system using pET vectors under the control of T7 promoter is generally recommended. The following protocol outlines a methodological approach:

• Clone the rlmE gene from D. vulgaris genomic DNA using PCR with high-fidelity polymerase.

• Insert the gene into pET vector with an N-terminal His-tag for easier purification.

• Transform the construct into E. coli BL21(DE3) or Rosetta(DE3) strains that provide tRNAs for rare codons.

• Induce protein expression with 0.5 mM IPTG at 18°C overnight to minimize inclusion body formation.

• Lyse cells and purify using Ni-NTA affinity chromatography followed by size exclusion chromatography.

This approach typically yields active enzyme suitable for in vitro studies of methyltransferase activity.

How does RlmE activity correlate with ribosome assembly defects in temperature-dependent studies?

RlmE activity shows a strong correlation with ribosome assembly efficiency, particularly at lower temperatures. Research on E. coli has demonstrated that rlmE knockout strains exhibit more severe growth and ribosome assembly defects at lower temperatures . This temperature-dependent phenomenon suggests that RlmE-mediated rRNA modification may provide structural stability to the ribosome that becomes more critical under cold stress conditions.

To study this correlation, researchers should examine ribosome assembly profiles using sucrose gradient ultracentrifugation at various growth temperatures (e.g., 20°C, 30°C, 37°C, and 42°C). A comprehensive analysis should include:

These values are estimates based on E. coli studies and should be determined experimentally for D. vulgaris.

What approaches can distinguish between the catalytic and structural roles of RlmE in ribosome assembly?

Distinguishing between catalytic and structural roles requires a multi-faceted experimental approach:

• Site-directed mutagenesis: Create catalytically inactive mutants by targeting residues in the S-adenosylmethionine (SAM) binding pocket or catalytic center (e.g., E198A mutation as used in RlmB studies) . Compare their ability to complement growth and ribosome assembly defects in ΔrlmE strains.

• Domain deletion/swapping: Engineer chimeric proteins where the methyltransferase domain is replaced with a homologous domain from another methyltransferase to test if the non-catalytic domains are sufficient for ribosome assembly.

• In vitro reconstitution: Use purified components to reconstitute ribosome assembly with:

  • Wild-type RlmE

  • Catalytically inactive RlmE

  • No RlmE

• Structural studies: Employ cryo-EM to visualize RlmE binding to ribosomal assembly intermediates, with and without SAM, to identify structural roles independent of methylation.

The results from these approaches should be integrated to develop a comprehensive model of RlmE's dual functionality.

How can we determine the interdependence between RlmE and other rRNA modification enzymes?

Research has shown that rRNA modification enzymes exhibit functional interdependence during ribosome assembly . To investigate these relationships for D. vulgaris RlmE:

• Generate multiple knockout strains lacking combinations of rRNA modification enzymes (e.g., ΔrlmE/ΔrlmB, ΔrlmE/ΔrlmKL, ΔrlmE/ΔrluC).

• Analyze ribosome assembly profiles for each strain under various conditions.

• Perform complementation studies with plasmid-expressed individual enzymes to restore function.

• Employ RNA-seq to map modification patterns in various knockout combinations.

• Use mass spectrometry to quantify modification levels in different genetic backgrounds.

A systematic approach as described above would reveal hierarchical relationships and cooperative functions among rRNA modification enzymes, similar to what has been observed in E. coli where multiple knockouts (e.g., ΔrluC, ΔrlmKL, ΔrlmN, ΔrlmM, ΔrluE) resulted in more severe phenotypes than individual knockouts .

What are the optimal conditions for assaying RlmE methyltransferase activity in vitro?

For optimal in vitro assessment of D. vulgaris RlmE activity, consider the following methodological approach:

Substrate preparation:

• Use either native 23S rRNA extracted from ΔrlmE strains or in vitro transcribed domain V of 23S rRNA

• Ensure proper RNA folding through controlled heating and cooling cycles

Reaction conditions:

Activity detection methods:

• Radiometric assay using ³H-labeled SAM

• LC-MS/MS to detect methylated versus unmethylated nucleosides

• Antibody-based detection of specific modifications

• RNA fingerprinting by primer extension

When developing the assay, include appropriate controls such as heat-inactivated enzyme, catalytically inactive mutants, and reactions without SAM or RNA substrate.

How should researchers design experiments to study the impact of D. vulgaris RlmE on ribosome function in heterologous systems?

When studying D. vulgaris RlmE in heterologous systems (e.g., E. coli), consider these experimental design principles:

• Complementation approach: Transform E. coli ΔrlmE strains with plasmids expressing:

  • D. vulgaris wild-type RlmE

  • D. vulgaris catalytically inactive RlmE

  • E. coli RlmE (positive control)

  • Empty vector (negative control)

• Expression control: Use inducible promoters with varying inducer concentrations to achieve physiologically relevant expression levels.

• Growth conditions: Test under various stressors (temperature, antibiotics, oxidative stress) to reveal condition-specific phenotypes.

• Ribosome profiling: Analyze translation efficiency and accuracy using ribosome profiling techniques.

• Cross-species differences: Account for potential incompatibilities between D. vulgaris RlmE and E. coli ribosomes by including domain-swapping experiments.

This approach allows for systematic assessment of functional conservation and divergence between RlmE orthologs.

How can researchers resolve contradictory results when studying RlmE function across different bacterial species?

When faced with contradictory results in cross-species RlmE studies, implement this systematic troubleshooting framework:

• Examine methodological differences:

  • Growth conditions (aerobic vs. anaerobic, temperature, media composition)

  • Protein expression levels (overexpression vs. native)

  • Assay conditions (in vitro vs. in vivo)

• Consider species-specific factors:

  • Evolutionary distance between species (e.g., E. coli vs. D. vulgaris)

  • Different ribosome assembly pathways

  • Presence of redundant enzymes or pathways

• Validation strategies:

  • Use multiple independent methods to confirm observations

  • Develop species-specific assays

  • Engineer hybrid ribosomes to pinpoint species-specific interactions

• Quantitative comparison:

  • Normalize data to appropriate references

  • Use statistical methods that account for species-specific variation

  • Consider kinetic differences rather than just endpoint measurements

When reporting results, clearly document all experimental conditions and discuss potential species-specific factors that might explain discrepancies with published literature.

What analytical approaches can detect subtle phenotypes in RlmE mutant strains?

Traditional growth rate measurements may miss subtle phenotypes in RlmE mutant strains. Consider these advanced analytical approaches:

• Competition assays: Co-culture wild-type and mutant strains to detect small fitness differences over multiple generations.

• Ribosome composition analysis:

  • Quantitative mass spectrometry to detect stoichiometric changes in ribosomal proteins

  • rRNA modification mapping using next-generation sequencing

  • Structure probing using chemical reagents (SHAPE, DMS)

• Translation fidelity assays:

  • Dual luciferase reporters with programmed errors

  • β-galactosidase readthrough assays

  • Mass spectrometry analysis of error frequency in the proteome

• Ribosome dynamics:

  • Single-molecule FRET to measure conformational changes

  • Ribosome profiling to assess translation elongation rates

  • tRNA binding and translocation kinetics

• Stress response quantification:

  • Transcriptomics to detect activation of stress pathways

  • Proteomics to identify compensatory mechanisms

  • Metabolomics to identify downstream metabolic effects

These approaches can reveal functional implications of RlmE that may not be apparent in standard growth assays.

What strategies overcome difficulties in expressing active recombinant D. vulgaris RlmE?

D. vulgaris proteins can present expression challenges due to codon bias and the anaerobic nature of this organism. Consider these methodological solutions:

• Codon optimization:

  • Redesign the gene sequence for optimal expression in E. coli

  • Use specialized strains containing rare tRNAs (e.g., Rosetta)

• Fusion partners to enhance solubility:

  • MBP (maltose-binding protein)

  • SUMO

  • Thioredoxin

  • Include removable tags via TEV or PreScission protease sites

• Expression conditions optimization:

  • Lower temperatures (16-20°C)

  • Reduced inducer concentration

  • Co-expression with chaperones

  • Anaerobic expression systems

• Refolding strategies for inclusion bodies:

  • Gradual dialysis

  • On-column refolding

  • Pulse dilution

• Alternative expression systems:

  • Cell-free protein synthesis

  • Bacillus subtilis

  • Anaerobic expression hosts

The table below summarizes reported success rates with different approaches:

How can researchers accurately assess the modification status of specific rRNA nucleotides?

Several methodological approaches can be employed to accurately determine the modification status of specific rRNA nucleotides targeted by RlmE:

• Reverse transcription-based methods:

  • Primer extension (modification causes RT stop or misincorporation)

  • SHAPE chemistry for structural impact of modifications

  • 2'-OH methylation detection using reduced dNTP concentration

• Mass spectrometry approaches:

  • LC-MS/MS of digested rRNA oligonucleotides

  • MALDI-TOF analysis of RNase digestion products

  • Comparative analysis between wild-type and mutant samples

• Chemical probing:

  • Selective reaction with unmethylated 2'-OH groups

  • Differential reactivity to DMS or CMCT

  • Orthogonal protection strategies

• Next-generation sequencing:

  • RNA-seq with specialized library preparation to detect modifications

  • Nanopore direct RNA sequencing which can detect modifications as altered current signals

  • TRIBE-seq for detecting modification-induced alterations

When implementing these methods, include appropriate controls:

• Unmodified in vitro transcribed rRNA

• rRNA from knockout strains lacking specific modification enzymes

• Synthetic oligonucleotides with defined modifications