Recombinant Thermus thermophilus 50S ribosomal protein L2 (rplB)

How does L2 interact with ribosomal RNA in the 50S subunit?

L2 makes extensive contacts with multiple domains of ribosomal RNA:

• The globular domain interacts primarily with helices 79 and 65

• The middle bridge region is embedded in rRNA, contacting helices 33, 65, 66, and 67

• The extension region closely approaches the peptidyl-tRNA and strongly interacts with the major groove of helix 93 near the peptidyl transferase center (PTC)

The primary L2-binding site has been biochemically characterized and maps to a highly conserved stem-loop structure of H66 (nucleotide positions 1792–1827) in domain IV of 23S rRNA. Specifically, the globular RNA-binding domain (RBD) of L2 binds an internal bulge structure (np. 1799–1800 and 1817–1820) of H66 .

These extensive RNA interactions explain why L2 is considered essential for ribosome assembly and function, with a large surface area displaying characteristics typical of RNA-binding proteins .

What experimental methods are used to study L2 and its role in ribosome function?

Several complementary methods are used to investigate L2's structure and function:

These methods often require careful experimental design with appropriate controls. For experiments involving active ribosomal particles, researchers typically use a between-subjects design comparing wild-type and mutant ribosomes, or a withdrawal design where L2 is selectively removed and then reconstituted .

What is the significance of L2 conservation across species?

L2 is one of the most highly conserved ribosomal proteins across all three domains of life (Bacteria, Archaea, and Eukarya):

• Due to its high degree of conservation, L2 is considered one of the most evolutionarily ancient ribosomal proteins

• Structural analysis suggests its RNA-binding domains have homology to common RNA/DNA-binding motifs found in many proteins (OB fold and SH3-like barrel)

• The conservation extends to its functional role in peptidyl transferase activity, suggesting it was part of the ancestral ribosome

Interestingly, experiments have shown that variants of the L2-binding site in H66 from eukaryotes (Class II binding sites) can be functional in bacterial ribosomes. For example, the sequence found in the African lungfish Protopterus aethiopicus can function in E. coli ribosomes . This finding demonstrates the evolutionary flexibility of this interaction despite its importance.

How do mutations in the L2 protein affect ribosome assembly and function?

Mutations in L2 can have distinct effects on ribosome function depending on their location within the protein structure:

Studies with yeast L2 (RPL2A) identified two classes of mutations with different functional impacts:

Class 1 Mutations (Globular Domain):

• V48D and L125Q mutations in the SH3 β-barrel domain

• Strongly affect ribosomal A-site associated functions

• Impair peptidyltransferase activity and subunit joining

• May affect interactions with Helix 55 and the Helix 65-66 structure

• Primarily cause 60S subunit biogenesis defects

Class 2 Mutations (Extension Domain):

• H215Y mutation at the tip of the extended domain

• Specifically affects peptidyl-tRNA binding and peptidyltransferase activity

• Interacts with Helix 93

Both classes affect rRNA structure far from the protein's location, suggesting L2 has allosteric effects on ribosome structure. The findings suggest some flexibility in L2's neck region between domains, which may help coordinate tRNA-ribosome interactions .

For designing mutation studies, researchers should consider:

• Using random mutagenesis libraries to identify functional domains

• Employing complementation assays with conditional lethal strains

• Analyzing effects on ribosome assembly using sucrose density gradient profiles

• Measuring specific ribosomal functions (subunit association, peptidyltransferase activity)

• Correlating functional defects with structural changes using cryo-EM

What methodological approaches can resolve contradictions in L2 localization within the ribosome?

Early studies presented an apparent contradiction regarding L2's location in the ribosome:

• L2 was detected on the cytoplasmic side of the 50S subunit

• Yet it could be labeled by erythromycin derivatives bound near the peptidyl-transfer center at the interface side

This contradiction was resolved through structural studies showing L2 has multiple domains that span different regions of the ribosome:

Recommended methodological approach:

• Multi-method structural analysis:

  • X-ray crystallography of isolated L2 to identify domains

  • Cryo-EM of intact ribosomes to visualize L2 in context

  • Cross-linking studies with labeled antibiotics or rRNA

• Domain mapping experiments:

  • Create truncated versions of L2 containing individual domains

  • Test each domain's interaction with rRNA and antibiotics

  • Use fluorescence resonance energy transfer (FRET) to measure distances

• Computational modeling:

  • Generate structural models incorporating all experimental data

  • Simulate dynamic movements of L2 domains during translation

The structural data revealed that L2 consists of a globular domain on the solvent-exposed side of the ribosome connected by a bridge region to an extension that approaches the peptidyl transferase center, thus explaining how a single protein can interact with both the exterior and the core functional regions of the ribosome .

How can researchers design experiments to analyze L2's role in erythromycin resistance?

Erythromycin resistance has been linked to mutations in L2, particularly in its protruding β hairpin region. To effectively study this relationship:

Experimental Design Approach:

• Site-directed mutagenesis:

  • Target the β hairpin region where erythromycin resistance mutations occur

  • Create a library of mutations in this region

  • Express mutant L2 proteins in cells and measure minimum inhibitory concentrations (MICs) for erythromycin

• Structural analysis:

  • Perform crystallography or cryo-EM on ribosomes containing resistant L2 variants

  • Compare with structures of wild-type ribosomes bound to erythromycin

  • Map changes in the drug binding pocket

• Functional assays:

  • Measure peptidyl transferase activity in the presence of erythromycin

  • Analyze translation of specific reporter mRNAs

  • Determine if resistance mechanisms involve altered drug binding or compensatory structural changes

• In vivo verification:

  • Use single-subject experimental design with reversal phases

  • Test each mutant in multiple antibiotic concentrations

  • Verify that phenotypes can be attributed to the L2 mutations by complementation

Current evidence suggests that erythromycin resistance mutations in L2 are located in the protruding β hairpin that interacts with rRNA near the peptidyl transferase center. This region might be directly involved in the erythromycin binding site, while the opposite end of L2 remains exposed to the cytoplasm .

What are the methodological considerations for studying L2's interaction with Helix 66 of 23S rRNA?

Helix 66 (H66) of 23S rRNA is a primary binding site for L2. Studies focusing on this interaction require careful experimental design:

Recommended experimental approach:

• Library construction and screening:

  • Generate a randomized library of the internal six nucleotides of the L2-binding site in H66

  • Use Systematic Evolution of Ligands by EXponential enrichment (SELEX) or functional genetic selection to identify viable variants

• Functional classification:

  • Categorize variants based on growth rates and ribosome assembly

  • Group sequences into classes (e.g., Class I vs. Class II binding sites)

  • Measure doubling times to quantify effects on cellular fitness

• Structural analysis:

  • Map interactions using chemical footprinting

  • Analyze base-triple formations critical for L2 binding

  • Identify co-variant positions indicating functional interactions

Studies have revealed two classes of L2-binding sites in H66:

• Class I (bacterial-type): Contains specific base-triple interactions

• Class II (eukaryotic-type): Contains an alternative base-triple configuration

Specific nucleotide positions show co-variation (e.g., G1817 and U1817 preferentially selected with C1800 and A1800, respectively), indicating functional interaction between these positions .

These findings demonstrate the importance of designing experiments that can detect compensatory mutations and functional interactions between nucleotides .

How can peptidyl transferase activity be experimentally attributed to L2 versus rRNA components?

The peptidyl transferase center (PTC) is now understood to be primarily composed of rRNA, yet proteins like L2 and L3 are required for efficient activity. Designing experiments to disentangle their contributions requires careful methodology:

Experimental approach:

• Subribosomal particle preparation:

  • Extract 50S subunits from thermophilic bacteria (e.g., T. aquaticus, T. thermophilus)

  • Use progressive extraction methods (protease digestion, SDS treatment, phenol extraction)

  • Isolate particles that retain peptidyl transferase activity

• Compositional analysis:

  • Characterize RNA content using gel electrophoresis

  • Identify remaining proteins using N-terminal sequencing

  • Quantify stoichiometry of remaining components

• Activity correlation:

  • Measure peptidyl transferase activity at each extraction step

  • Correlate activity loss with removal of specific components

  • Reconstitute particles with purified components to restore activity

Research with T. aquaticus has shown that active subribosomal particles contained:

• 23S and 5S rRNA

• Eight ribosomal proteins in notable amounts: L2, L3, L13, L15, L17, L18, L21, and L22

• Near-stoichiometric levels of L2, L3, L13, and L22

Complete removal of all proteins resulted in particle unfolding and loss of activity, while RNase treatment increased the accessibility of remaining proteins to protease digestion. These findings suggest a structural organization where an RNA "cage" surrounds a core of ribosomal proteins, with L2 and L3 being essential components for peptidyl transferase activity .

What are the best practices for expressing and purifying recombinant T. thermophilus L2 for structural studies?

For researchers needing high-quality recombinant L2 protein for structural or functional studies:

Recommended protocol:

• Expression system selection:

  • E. coli is the preferred expression system for T. thermophilus L2

  • Use a vector with an inducible promoter (T7 or tac)

  • Consider adding purification tags (His-tag most common)

• Expression optimization:

  • Induce at OD600 0.6-0.8

  • Express at 30°C for 4-6 hours or 18°C overnight

  • Include protease inhibitors during lysis

• Purification strategy:

  • Use immobilized metal affinity chromatography (IMAC) for His-tagged protein

  • Follow with size exclusion chromatography for higher purity

  • Aim for >85% purity by SDS-PAGE

• Storage recommendations:

  • Reconstitute lyophilized protein in deionized sterile water to 0.1-1.0 mg/mL

  • Add glycerol to 5-50% final concentration (50% recommended)

  • Make small aliquots and store at -20°C/-80°C

  • Avoid repeated freeze-thaw cycles; use working aliquots at 4°C for up to one week

• Quality control:

  • Verify sequence by mass spectrometry

  • Check folding by circular dichroism

  • Test RNA-binding activity prior to structural studies

The shelf-life of properly stored L2 is typically 6 months for liquid formulations and 12 months for lyophilized preparations at -20°C/-80°C .