Recombinant Methanococcus maripaludis 30S ribosomal protein S15P/S13e (rps15p)

What is the role of 30S ribosomal protein S15P/S13e in ribosome assembly?

S15P/S13e serves as a primary binding protein that organizes the central domain of 16S rRNA in the 30S ribosomal subunit of M. maripaludis. Similar to its homolog in Escherichia coli, it likely plays a critical role in the hierarchical assembly of the 30S subunit platform. In E. coli, S15 orchestrates the assembly of ribosomal proteins S6, S11, S18, and S21 with the central domain of 16S rRNA . The protein binds directly to 16S rRNA, creating a structured environment that allows secondary and tertiary binding proteins to associate with the emerging complex. As an archaeal organism, M. maripaludis likely shows some differences in assembly pathways, but the fundamental role of S15P/S13e in organizing the platform region is presumably conserved.

How does the function of S15P/S13e in M. maripaludis compare to bacterial homologs?

While both archaeal S15P/S13e and bacterial S15 serve as primary binding proteins in their respective ribosomes, there are notable functional differences. The archaeal protein often demonstrates greater thermostability, reflecting the extremophilic nature of many archaeal species. In E. coli, studies have shown that deletion of the gene encoding S15 (rpsO) creates viable but growth-compromised strains, demonstrating surprising plasticity in the in vivo assembly process . This plasticity may be less pronounced in archaeal systems like M. maripaludis, which have evolved under different selective pressures. Additionally, unlike bacterial S15, archaeal S15P/S13e may participate in archaeal-specific bridge connections between ribosomal subunits that reflect the evolutionary relationship between archaeal and eukaryotic translation systems.

What expression systems are most effective for producing recombinant M. maripaludis S15P/S13e?

• E. coli expression systems: Despite potential codon usage differences, optimized E. coli expression systems with archaeal-friendly codon optimization can yield functional protein.

• Homologous expression: The recently developed CRISPR/Cas12a genome editing toolbox for M. maripaludis enables direct manipulation of the native organism for homologous expression. This system has demonstrated high efficiency with positive editing rates of approximately 95% .

• Cell-free systems: For difficult-to-express proteins, archaeal cell-free translation systems may preserve native folding and post-translational modifications.

When using heterologous expression, researchers should include appropriate affinity tags (His6, Strep-tag II) while considering their potential impact on protein structure and function.

What purification strategies yield the highest purity and activity for functional studies?

A multi-step purification approach is recommended for isolating high-purity, functionally active S15P/S13e:

• Initial capture: Immobilized metal affinity chromatography (IMAC) using Ni-NTA or Co-NTA resins for His-tagged constructs.

• Intermediate purification: Ion exchange chromatography using a salt gradient (typically 0-1M NaCl).

• Polishing step: Size exclusion chromatography to remove aggregates and obtain homogeneous protein.

Critical buffer considerations include:

• Maintaining pH 7.0-8.0

• Including 5-10% glycerol to enhance stability

• Adding reducing agents (1-5 mM DTT or β-mercaptoethanol) to prevent oxidation of cysteine residues

• Including RNase inhibitors if co-purifying with rRNA fragments

For activity assays, maintaining the protein in buffers that mimic the intracellular environment of M. maripaludis is crucial for preserving native structure and function.

How can CRISPR/Cas12a technology be used to study S15P/S13e function in M. maripaludis?

The recently developed CRISPR/Cas12a genome editing toolbox for M. maripaludis provides a powerful approach for studying S15P/S13e function. This system has demonstrated remarkable efficiency with positive editing rates of approximately 95% . Researchers can utilize this technique through several strategic approaches:

• Gene knockouts: Creating a complete deletion of the gene encoding S15P/S13e to assess viability and growth phenotypes under various conditions.

• Domain mutations: Introducing specific mutations in functional domains to assess their impact on ribosome assembly and function.

• Promoter swapping: Replacing the native promoter with constitutive or inducible promoters to control expression levels. Several characterized promoters in M. maripaludis show differential strength, including PmcrR_JJ, Pfla_JJ, and PglnA .

• Epitope tagging: Introducing tags for immunoprecipitation or fluorescent proteins for localization studies.

The general procedure involves:

• Designing gRNAs targeting the gene of interest

• Constructing repair templates with desired modifications

• Co-transformation of the CRISPR/Cas12a plasmid and repair template

• Selection and verification of edited strains

The CRISPR/Cas12a system requires only a single round of homologous recombination and avoids merodiploid formation, increasing efficiency compared to traditional methods .

What phenotypic changes can be expected in S15P/S13e deletion or mutation strains?

Based on studies of S15 deletion in E. coli, researchers can anticipate several phenotypic consequences when manipulating S15P/S13e in M. maripaludis:

• Growth impairment: Strains lacking S15P/S13e likely exhibit extended doubling times compared to wild-type strains, especially at optimal growth temperatures .

• Cold sensitivity: Deletion strains may show pronounced growth defects at reduced temperatures, reflecting the critical role of S15P/S13e in ribosome assembly under suboptimal conditions .

• Ribosome profile alterations: Polysome profile analysis would likely reveal abnormal subunit ratios, with potential accumulation of precursor particles and reduced 70S ribosome formation .

• rRNA processing defects: As observed in E. coli Δrps0 strains, M. maripaludis lacking S15P/S13e may accumulate 16S rRNA precursors with improper 5' end processing .

• Subunit association defects: 30S subunits lacking S15P/S13e may show reduced capacity to form stable associations with 50S subunits in vitro and in vivo .

The severity of these phenotypes may vary depending on growth conditions, with more pronounced effects under stress conditions such as temperature shifts or nutrient limitation.

What techniques are most informative for studying S15P/S13e interactions with rRNA?

Several complementary approaches provide insights into S15P/S13e-rRNA interactions:

• Cryo-electron microscopy (cryo-EM): High-resolution structural determination of intact ribosomes, allowing visualization of S15P/S13e in its native context. Modern cryo-EM can achieve near-atomic resolution, revealing specific protein-RNA contacts.

• X-ray crystallography: For studying isolated S15P/S13e in complex with specific rRNA fragments.

• Nuclear Magnetic Resonance (NMR): Particularly useful for studying dynamic interactions between S15P/S13e and rRNA fragments, providing information about conformational changes.

• Chemical probing techniques:

  • SHAPE (Selective 2'-hydroxyl acylation analyzed by primer extension)

  • DMS (dimethyl sulfate) probing

  • CMCT (1-cyclohexyl-3-(2-morpholinoethyl)carbodiimide metho-p-toluenesulfonate) modification
    These methods reveal changes in RNA structure upon protein binding.

• Isothermal Titration Calorimetry (ITC): Quantitative measurement of binding affinity and thermodynamic parameters of S15P/S13e-rRNA interactions. Similar studies with E. coli S15 have provided valuable insights into assembly dependencies .

• Fluorescence techniques: Including FRET (Förster Resonance Energy Transfer) to monitor conformational changes during assembly.

How can researchers distinguish between direct and indirect effects of S15P/S13e on ribosome assembly?

Distinguishing direct from indirect effects requires a multi-faceted experimental approach:

• In vitro reconstitution assays: Systematically omitting or adding S15P/S13e to reconstitution reactions to identify direct dependencies. This approach revealed the critical role of S15 in E. coli ribosome assembly in vitro .

• Time-resolved assembly maps: Using pulse-labeling techniques to track the temporal sequence of protein binding during ribosome assembly.

• Crosslinking studies: Employing UV or chemical crosslinking to capture direct interaction partners of S15P/S13e.

• Compensatory mutation analysis: Introducing mutations in potential binding partners and assessing whether complementary mutations in S15P/S13e can restore function.

• Comparative analysis: Between in vitro and in vivo assembly. E. coli studies have revealed surprising differences, with in vivo assembly showing greater plasticity than predicted by in vitro studies .

• Assembly intermediates characterization: Isolating assembly intermediates from cells with depleted or mutated S15P/S13e and determining their composition and structure.

• Quantitative mass spectrometry: Using SILAC (Stable Isotope Labeling with Amino acids in Cell culture) or TMT (Tandem Mass Tag) approaches to quantify changes in protein association during assembly.

How does temperature affect S15P/S13e function in ribosome assembly?

Temperature significantly impacts S15P/S13e function in ribosome assembly, with important implications for archaeal biology:

• Cold sensitivity: Based on E. coli studies, deletion of S15 leads to cold-sensitive phenotypes, indicating that S15 becomes critical for assembly at lower temperatures . In M. maripaludis, which normally grows at moderate temperatures (35-40°C), S15P/S13e may play a similarly critical role at the lower end of its temperature range.

• Assembly kinetics: Temperature directly affects the kinetics of ribosome assembly, with lower temperatures potentially revealing rate-limiting steps that are dependent on S15P/S13e.

• Structural implications: At lower temperatures, increased RNA secondary structure stability may require more active participation of S15P/S13e in promoting proper folding of the 16S rRNA central domain.

• Conformational changes: Temperature-dependent conformational changes in S15P/S13e may affect its binding affinity for rRNA and neighboring proteins.

• Compensatory mechanisms: Alternative assembly pathways that bypass the need for S15P/S13e may become less efficient at lower temperatures, explaining the cold-sensitive phenotype observed in deletion strains .

Research approach should include growth rate measurements, ribosome profiling, and in vitro assembly assays conducted across a temperature gradient to fully characterize these effects.

What is the plasticity of ribosome assembly pathways in the absence of S15P/S13e in M. maripaludis?

Studies in E. coli have revealed surprising plasticity in ribosome assembly in the absence of S15, which challenges the strict hierarchical model established through in vitro studies . For M. maripaludis:

• Alternative assembly pathways: The ability of M. maripaludis to potentially assemble functional ribosomes in the absence of S15P/S13e would indicate flexible assembly pathways not predicted by in vitro systems.

• Binding dependencies: While in vitro studies in E. coli suggest that S6/S18 heterodimer cannot bind to 16S rRNA without prior S15 binding , in vivo systems may employ additional factors or conditions that permit this binding.

• Compensatory mechanisms: Other ribosomal proteins or assembly factors may adopt compensatory roles in the absence of S15P/S13e, particularly under optimal growth conditions.

• Quantitative assessment: Ribosome composition analysis of particles formed in S15P/S13e deletion strains would reveal whether all other platform proteins (homologs of S6, S11, S18, and S21) are incorporated despite S15P/S13e absence, as observed in E. coli .

• Evolutionary implications: The plasticity observed suggests evolutionary adaptation to ensure ribosome assembly can proceed even when certain components are unavailable, potentially reflecting selective pressures in archaeal evolution.

To investigate this plasticity, researchers should combine genetic approaches (deletion strains) with quantitative proteomic analysis of ribosomal particles and functional assays of translation activity.