Recombinant Mesoplasma florum 50S ribosomal protein L14 (rplN)

What are the optimal conditions for reconstituting recombinant M. florum rplN for functional studies?

For optimal reconstitution of lyophilized recombinant M. florum rplN:

• Centrifuge the vial briefly before opening to ensure the protein is at the bottom

• Reconstitute in deionized sterile water to a concentration of 0.1-1.0 mg/mL

• Add glycerol to a final concentration of 5-50% (50% is recommended) for long-term storage

• Aliquot the reconstituted protein to minimize freeze-thaw cycles

• Store working aliquots at 4°C for up to one week

• For longer storage, keep at -20°C/-80°C

Important note: Repeated freezing and thawing significantly reduces protein activity and should be avoided. The shelf life of the liquid form is approximately 6 months at -20°C/-80°C, while the lyophilized form remains stable for about 12 months at the same temperature .

How can crosslinking experiments be designed to study M. florum rplN interactions within the ribosome?

To investigate protein-protein interactions involving rplN within the M. florum ribosome:

• Whole-cell crosslinking approach:

  • Culture M. florum cells to mid-exponential phase (OD measurements at 560 nm)

  • Harvest cells and resuspend in an appropriate buffer

  • Apply a protein crosslinker (such as formaldehyde or DSS)

  • Lyse cells and isolate ribosomes through ultracentrifugation

  • Digest with proteases and analyze crosslinked peptides by MS/MS

• RNA-protein crosslinking:

  • UV-irradiate whole cells (254 nm) to capture RNA-protein interactions

  • Process samples with TRIzol™ reagent for extraction

  • Analyze using the RNPxl pipeline to identify crosslinked peptides

This methodology has been successfully applied in minimal cells to map ribosomal protein interaction networks and can reveal how rplN connects with other ribosomal components and potential regulatory factors.

What role does rplN play in the architecture of the minimal M. florum ribosome?

The 50S ribosomal protein L14 (rplN) in M. florum serves critical structural and functional roles:

• Structural integrity: Acts as a central architectural component of the 50S ribosomal subunit, forming important bridges that maintain the three-dimensional structure of the large subunit.

• rRNA interactions: Forms multiple contacts with rRNA molecules, particularly in domains I and V of the 23S rRNA, stabilizing their tertiary structure.

• Intersubunit bridge formation: Participates in the formation of bridges between the 30S and 50S subunits during translation.

• Peptidyl transferase center proximity: Positioned near the peptidyl transferase center, potentially influencing the core catalytic function of the ribosome.

These roles make rplN essential in minimal genome organisms like M. florum, where the protein synthesis machinery has been streamlined through evolutionary reduction. Studies of the "expressome" (the coupled transcription-translation machinery) in minimal cells suggest that ribosomal proteins like L14 may have additional roles in coordinating these processes through protein-protein interactions with other cellular components .

How does the expression level of rplN compare to other ribosomal proteins in M. florum?

Transcriptome and proteome analysis of M. florum reveals important insights about the expression patterns of ribosomal proteins:

• Transcription units: The rplN gene is part of a complex transcription unit architecture with overlapping transcription units, suggesting sophisticated regulation mechanisms despite genome minimization.

• Promoter organization: Transcriptome profiling has identified a conserved promoter motif associated with ribosomal protein genes in M. florum, including those in the rplN operon.

• Absolute abundance: Through biomass quantification experiments, researchers have estimated the absolute molecular abundance of ribosomal proteins, with rplN present at levels proportional to its stoichiometric requirements in assembled ribosomes.

• Coordinated expression: The transcription and protein expression levels of all ribosomal proteins, including rplN, show coordinated regulation to ensure proper ribosome assembly despite the minimized genome .

This expression pattern underscores the importance of maintaining proper stoichiometry in the minimal translation machinery of M. florum.

Why is rplN considered essential in minimal genome studies of M. florum?

The rplN gene has been identified as essential in M. florum through several lines of evidence:

• Transposon mutagenesis: Studies using Tn5 transposon insertion libraries in M. florum have demonstrated that disruption of rplN is lethal, confirming its essentiality .

• Comparative genomics: Analysis of 13 M. florum strains collected from diverse environments shows conservation of rplN across all strains, further supporting its essentiality .

• Minimal genome design: In computational predictions for minimal genome design in M. florum, rplN has consistently been retained due to its critical role in ribosome assembly and function.

• Evolutionary conservation: The high degree of sequence conservation of rplN among different strains of M. florum and related species suggests strong selective pressure to maintain its function .

These findings collectively establish rplN as a core component of the minimal genome necessary for cellular viability, making it an important protein for synthetic biology efforts aimed at creating minimal cells.

How does M. florum rplN compare to its counterpart in other minimal genome organisms?

The high degree of conservation in rplN across these minimal and near-minimal organisms highlights its fundamental importance to ribosome function even as other cellular systems have been reduced or eliminated through evolution or synthetic reduction . Notable differences in sequence may reflect adaptations to specific growth environments or compensatory changes to maintain proper ribosome structure and function despite other genomic alterations.

How can M. florum rplN be used in studies of ribosome assembly pathways in minimal cells?

Recombinant M. florum rplN provides a valuable tool for investigating ribosome assembly in minimal cells through several experimental approaches:

• In vitro ribosome reconstitution:

  • Use purified recombinant rplN along with other ribosomal proteins and rRNA to reconstruct minimal functional ribosomes

  • Monitor assembly intermediates using techniques like sucrose gradient ultracentrifugation, cryo-EM, or chemical probing

  • Determine the order and kinetics of assembly by time-course experiments with labeled components

• Assembly factor identification:

  • Perform pull-down experiments using tagged recombinant rplN to identify potential assembly factors in M. florum lysates

  • Compare assembly factor requirements between M. florum and more complex organisms to understand the minimal requirements for ribosome biogenesis

• Mutational analysis:

  • Introduce specific mutations in recombinant rplN to study their effects on ribosome assembly and function

  • Identify critical residues by complementation experiments in conditionally depleted strains

These approaches can reveal whether minimal cells like M. florum use simplified assembly pathways compared to more complex organisms, potentially identifying core assembly mechanisms that have been conserved throughout evolution .

What can we learn about minimal translation systems by studying interactions between M. florum rplN and antibiotic compounds?

Studying interactions between M. florum rplN and antibiotics can provide valuable insights into minimal translation systems:

• Binding site analysis:

  • Many antibiotics target the large ribosomal subunit where L14 is located

  • Comparing antibiotic binding sites between M. florum and other organisms can reveal conserved functional regions

  • The minimal nature of M. florum may expose core antibiotic resistance or sensitivity determinants

• Resistance mechanisms:

  • Generate point mutations in recombinant rplN to study their effects on antibiotic sensitivity

  • Examine whether M. florum's natural resistance or sensitivity to specific antibiotics correlates with sequence variations in rplN

• Methodological approach:

  • Express recombinant wild-type and mutant versions of M. florum rplN

  • Reconstitute ribosomes with these variants

  • Perform in vitro translation assays in the presence of various antibiotics

  • Use structural techniques (X-ray crystallography, cryo-EM) to visualize antibiotic binding sites

This research could help identify the minimal structural requirements for antibiotic resistance, potentially guiding the development of new antimicrobial compounds that target essential features of the bacterial ribosome .

How might CRISPR-based genome editing of rplN in M. florum advance minimal genome research?

CRISPR-based genome editing of rplN in M. florum presents several promising research avenues:

• Minimal functional domains:

  • Create precise deletions or mutations to identify the minimal functional domains of rplN

  • Determine which regions can be modified without losing viability

  • Map the essential interaction surfaces of the protein

• Codon optimization studies:

  • Recode the rplN gene with alternative codons while maintaining amino acid sequence

  • Study effects on translation efficiency and growth rates

  • Identify optimal codon usage for minimal genomes

• Implementation strategy:

  • Design sgRNAs targeting specific regions of the rplN gene

  • Use CRISPR/Cas9 or other CRISPR systems (potentially with reduced off-target effects)

  • Incorporate homology-directed repair templates for precise editing

  • Screen edited strains for viability and growth characteristics

  • Analyze ribosome assembly and function in successful mutants

This approach could overcome historical challenges in genetic manipulation of mycoplasma species, as traditional approaches like transposon mutagenesis provide limited precision. Recent advances in CRISPR-based tools for mycoplasmas make these experiments increasingly feasible .

How could ribosome profiling experiments utilizing M. florum rplN advance our understanding of translation in minimal cells?

Ribosome profiling experiments focused on rplN could provide unprecedented insights into translation in minimal cells:

• Experimental design:

  • Culture M. florum under various conditions

  • Harvest cells and isolate ribosome-protected mRNA fragments

  • Sequence these fragments to determine ribosome positions on mRNAs

  • Compare results with transcriptome and proteome data to assess translation efficiency

• Key research questions:

  • How does translation efficiency vary across the minimal genome?

  • Are there specific features of mRNAs that influence translation in this minimal organism?

  • How does the limited tRNA set of M. florum affect codon usage and translation speed?

  • Are there differences in translational pausing compared to more complex organisms?

• Integration with growth kinetics:

  • M. florum has a rapid doubling time of ~32 minutes at its optimal growth temperature of 34°C

  • Ribosome profiling at different growth phases could reveal how translation dynamics change throughout the cell cycle in a minimal organism

This approach could identify fundamental principles of translation that are conserved in minimal organisms and potentially illuminate evolutionary constraints on the translation apparatus.