Recombinant Enterococcus faecalis 30S ribosomal protein S14 2 (rpsN2)

What is the role of S14 protein in the Enterococcus faecalis ribosome?

Ribosomal protein S14 (encoded by rpsN2) is a crucial component of the 30S small ribosomal subunit in Enterococcus faecalis. It forms part of the intersubunit bridge B1c, establishing contacts with the large ribosomal subunit protein bL31. Specifically, S14 (uS14) interacts with bL31 along with uS19 and 16S nucleotides A1311 and G1312 in helix 42 . These interactions are essential for maintaining ribosomal stability during the rotational movements that occur during protein synthesis.

How conserved is the S14 protein structure across bacterial species?

The S14 protein structure shows varying degrees of conservation across bacterial species. While functional centers of the ribosome including antibiotic binding sites are generally well-conserved, peripheral regions show more structural differences. Comparing Enterococcus faecalis with model organisms like Escherichia coli (which has 76% sequence identity for 16S rRNA) and the more closely related Staphylococcus aureus reveals that while core functional elements remain highly conserved, species-specific structural differences do exist in peripheral regions .

What are the key structural features of the E. faecalis 30S ribosomal subunit?

The E. faecalis 30S ribosomal subunit exhibits dynamic conformational changes during protein synthesis. Key features include:

Even in the conformation closest to the classical state (class 4), the 30S body is rotated by 3.3°, while the 30S head domain shows significant rotation in four of five observed classes (15.5° to 19.7°) .

What is the recommended experimental design for studying rpsN2 function?

When designing experiments to study recombinant E. faecalis rpsN2 function, consider the following five-step approach:

• Define your variables clearly:

  • Independent variable: E. faecalis rpsN2 expression levels or specific mutations

  • Dependent variable: Ribosomal assembly efficiency, translation rates, or antibiotic susceptibility

  • Control for extraneous variables such as growth conditions and strain background

• Formulate a specific, testable hypothesis about rpsN2 function

• Design experimental treatments to manipulate rpsN2 expression or structure:

  • Gene knockout or knockdown

  • Site-directed mutagenesis

  • Complementation experiments

• Assign experimental subjects to treatment groups using either:

  • Between-subjects design: Different bacterial cultures receive different treatments

  • Within-subjects design: Same bacterial culture measured before and after treatment

• Plan measurement techniques for your dependent variables, such as:

  • Ribosome profiling

  • Cryo-electron microscopy

  • In vitro translation assays

  • Antibiotic susceptibility tests

How can I express and purify recombinant E. faecalis rpsN2 for structural studies?

For optimal expression and purification of recombinant E. faecalis rpsN2:

• Expression system selection: E. coli BL21(DE3) is recommended for ribosomal protein expression to avoid toxicity issues that might occur in the native host.

• Vector design: Create a construct with:

  • An N-terminal 6×His-tag for purification

  • A TEV protease cleavage site for tag removal

  • Codon optimization for E. coli if necessary

• Expression conditions:

  • Culture in LB medium at 37°C until OD600 reaches 0.6-0.8

  • Induce with 0.5-1 mM IPTG

  • Shift to 18-25°C for overnight expression to enhance solubility

• Purification protocol:

  • Lyse cells using sonication in buffer containing 50 mM Tris-HCl pH 8.0, 300 mM NaCl, 10 mM imidazole

  • Perform Ni-NTA affinity chromatography

  • Remove His-tag using TEV protease

  • Further purify using ion-exchange chromatography

  • Perform final polishing with size-exclusion chromatography

• Quality control:

  • Verify protein purity using SDS-PAGE (expected size approximately 10-12 kDa)

  • Confirm identity with Western blotting and/or mass spectrometry

  • Assess proper folding with circular dichroism spectroscopy

How does S14 contribute to ribosomal rotation states in E. faecalis?

S14 plays a critical role in maintaining intersubunit bridges during the various rotation states observed in the E. faecalis ribosome. Cryo-EM studies of the E. faecalis 70S ribosome have identified five distinct conformational classes with varying degrees of 30S body and head rotation.

In these structures, S14 (uS14) interacts with the C-terminal domain of bL31, which extends from the large subunit toward the small subunit. These interactions form part of intersubunit bridge B1c, which must be maintained throughout the rotational movements of the ribosome. The contacts between bL31 and S14, along with uS19 and 16S rRNA nucleotides A1311 and G1312 in helix 42, provide flexibility while maintaining the structural integrity of the ribosome .

The ability of S14 to maintain these contacts throughout different rotation states is essential for proper ribosome function, particularly during translation elongation when the ribosome cycles through different conformational states.

What are the implications of S14 conservation for antibiotic development against E. faecalis?

The conservation of ribosomal protein structures, including S14, has significant implications for antibiotic development against E. faecalis, an organism increasingly associated with antibiotic resistance. The high-resolution structure of the E. faecalis ribosome reveals that:

• Functional centers of the ribosome, including antibiotic binding sites, are strongly conserved between E. faecalis and other bacterial species

• Binding sites for clinically relevant antibiotics such as gentamicin, streptomycin, linezolid, quinupristin-dalfopristin, doxycycline, and tigecycline show high structural similarity between E. faecalis and E. coli

• Specific differences in nucleotide identity may affect antibiotic binding, such as the replacement of U1196 with adenine in the tigecycline binding site of E. faecalis

This conservation suggests that existing ribosome-targeting antibiotics should maintain efficacy against E. faecalis, but the specific structural differences might explain variations in susceptibility and could inform the development of optimized antibiotics with enhanced activity against enterococci.

How can Grad-seq be used to study rpsN2 interactions in E. faecalis?

Gradient profiling by sequencing (Grad-seq) offers a powerful approach to comprehensively study RNA-protein complexes in E. faecalis, including those involving rpsN2:

• Methodology:

  • Bacterial cultures are lysed under conditions that preserve native complexes

  • Lysates are fractionated by glycerol gradient ultracentrifugation

  • Fractions are analyzed for RNA content by RNA-seq and for protein content by mass spectrometry

  • Sedimentation profiles for RNAs and proteins are generated based on their distribution across fractions

• Application to rpsN2 research:

  • Identify RNA partners of S14 protein by comparing their sedimentation profiles

  • Discover previously unknown interactions between S14 and regulatory RNAs

  • Compare wild-type and mutant S14 sedimentation profiles to identify functional changes

• Advantages for E. faecalis research:

  • Provides a global view of RNA and protein complexes

  • Helps identify novel small RNAs (sRNAs) that might interact with ribosomal proteins

  • Enables comparison of complex formation between different conditions (e.g., antibiotic exposure)

How do mutations in rpsN2 affect ribosome assembly and antibiotic resistance?

Mutations in rpsN2 can significantly impact ribosome assembly and potentially contribute to antibiotic resistance through several mechanisms:

• Effects on ribosome assembly:

  • Mutations in ribosomal proteins often lead to assembly defects

  • Since S14 forms critical intersubunit bridges, mutations may affect the stability of the 30S-50S interface

  • Altered interactions with 16S rRNA could affect the folding and maturation of the small subunit

• Implications for antibiotic resistance:

  • Mutations near antibiotic binding sites can directly reduce drug affinity

  • Structural alterations can indirectly affect the binding of antibiotics that target the ribosome

  • Changes in intersubunit rotation dynamics may affect the action of antibiotics that target translocation

• Experimental approach to study mutations:

  • Generate site-directed mutations in recombinant rpsN2

  • Express mutant proteins in E. faecalis using allelic replacement

  • Assess ribosome assembly by sucrose gradient centrifugation

  • Measure antibiotic susceptibility using minimum inhibitory concentration (MIC) assays

  • Analyze structural changes using cryo-EM or other structural techniques

What role does S14 play in tRNA positioning during translation?

S14 contributes significantly to tRNA positioning during translation in E. faecalis. Cryo-EM studies have revealed that:

• In one of the rotational states of the E. faecalis ribosome (class 1), where the 30S body is rotated by 2° and the 30S head is rotated by 19.7°, a tRNA was observed in a chimeric pe/E position rather than the classical E site

• This chimeric tRNA position appears similar to that observed in Thermus thermophilus ribosomes with similar degrees of 30S head and body rotation

• S14, as part of the 30S head domain, moves significantly during these rotational movements, affecting the environment around the tRNA binding sites

• The interactions between S14 and large subunit components like bL31 help maintain the structural integrity of the ribosome during these dynamic states

These findings suggest that S14 plays an important role in facilitating the movement of tRNAs through the ribosome during translation, particularly in the E site region during translocation.

How do the structural differences in E. faecalis S14 compared to E. coli affect ribosome function?

The specific differences in S14 structure and its interactions may contribute to the unique biology of E. faecalis, including its pathogenicity and response to environmental stresses.