Recombinant 60S ribosomal protein L27a (rpl-27a)

What is the molecular structure and basic properties of RPL27A?

RPL27A is a component of the large ribosomal subunit (60S in eukaryotes) belonging to the universal ribosomal protein uL15 family. In humans, RPL27A spans amino acids 2-148 and is expressed as a recombinant protein with >95% purity in E. coli systems . The Drosophila melanogaster homolog has 149 amino acids with a molecular weight of 17,123 Daltons . RPL27A functions as part of the ribonucleoprotein complex responsible for protein synthesis in the cell . The protein undergoes post-translational modification, specifically hydroxylation on His-39 by the enzyme MINA .

How is the RPL27A gene organized across species?

The RPL27A gene structure shows notable conservation with some species-specific characteristics. In Drosophila melanogaster, hybridization studies with cDNA to polytene chromosomes indicate a single gene located on chromosome 3R at position 87F/88A . The gene produces a single mRNA transcript of approximately 650 nucleotides in length . Comparative genomic analysis reveals homology between Drosophila RPL27A and its counterparts in rat and yeast, as well as other members of the L15 ribosomal protein family, suggesting evolutionary conservation of this protein's function .

What functional domains characterize the RPL27A protein?

While the search results don't explicitly detail all functional domains of RPL27A, we can infer from homology studies that RPL27A contains domains critical for ribosome assembly and function. Interestingly, Drosophila RPL27A shows significant homology not only with ribosomal proteins but also with an invertebrate motor protein and a Drosophila photoreceptor morphogenesis protein . This suggests potential structural features that may be shared across diverse protein families. By analyzing related ribosomal proteins like L27 in E. coli, we can deduce that RPL27A likely contains domains that facilitate interaction with rRNA and positioning near the peptidyltransferase center of the ribosome .

What are the optimal conditions for recombinant RPL27A expression?

Recombinant RPL27A is typically expressed in Escherichia coli expression systems with a fusion tag to facilitate purification . Based on commercial production protocols, RPL27A can be expressed with an N-terminal His6-ABP tag (where ABP represents Albumin Binding Protein derived from Streptococcal Protein G) . The protein is commonly expressed as a full-length construct spanning amino acids 2-148 in humans . For expression optimization, researchers should consider:

• Expression vector selection with strong promoters (like T7)

• Codon optimization for E. coli if needed

• Induction conditions (temperature, IPTG concentration)

• Cell lysis conditions to preserve protein integrity

Drawing from protocols used for related ribosomal proteins, expression at lower temperatures (16-25°C) may improve solubility and proper folding .

What purification strategies yield the highest purity recombinant RPL27A?

High-purity recombinant RPL27A (>95%) can be achieved through optimized purification strategies . The primary method involves Immobilized Metal Affinity Chromatography (IMAC) for His-tagged constructs . The expected concentration after purification is greater than 0.5 mg/ml . For research requiring exceptionally pure preparations, consider this multi-step approach:

• IMAC using Ni-NTA or similar resins for initial capture

• Ion exchange chromatography as a secondary purification step

• Size exclusion chromatography for final polishing and buffer exchange

• Quality control using SDS-PAGE and Western blotting to confirm identity and purity

For structural studies, additional steps to remove potential aggregates or misfolded proteins may be necessary.

How can researchers assess the functional integrity of purified RPL27A?

Functional assessment of RPL27A should examine both its structural integrity and ability to participate in ribosome assembly and function. Drawing from methodologies used for ribosomal proteins, the following approaches are recommended:

• In vitro ribosome assembly assays: Monitoring the incorporation of RPL27A into ribosomal subunits using density gradient centrifugation .

• Translation activity assays: Using cell-free translation systems like the integrated synthesis, assembly and translation (iSAT) platform to assess how RPL27A variants affect protein synthesis .

• Binding assays: Evaluating interactions with rRNA and other ribosomal proteins using techniques such as surface plasmon resonance or pull-down assays.

• Structural analysis: Circular dichroism or nuclear magnetic resonance spectroscopy to assess proper folding, as demonstrated with L27 proteins .

What is the specific role of RPL27A in ribosome assembly?

While the search results don't directly address RPL27A's role in assembly, valuable insights can be drawn from studies of the related protein L27 in E. coli. When L27 is deleted, assembly of the 50S ribosomal subunit is severely perturbed, resulting in the accumulation of a 40S precursor particle that is also deficient in proteins L16, L20, and L21 . By analogy, RPL27A likely plays a critical role in the assembly of the eukaryotic 60S subunit.

The assembly process appears to follow an ordered pathway where RPL27A incorporation may be required for subsequent binding of other ribosomal proteins. Studies using density gradient centrifugation have shown that mutations in critical ribosomal components significantly alter the distribution between free subunits and fully assembled ribosomes . For instance, one study demonstrated that compared to wild-type ribosomes (with a relative ratio of subunits to 70S + polysomes of ~1.4), certain mutations caused an ~11-fold greater population of individual subunits .

How does RPL27A contribute to peptidyltransferase activity?

Drawing from studies on E. coli L27, RPL27A likely plays an important role in peptidyltransferase activity. In E. coli, ribosomes lacking L27 are impaired in peptidyltransferase activity, most likely due to a defect in the binding of aminoacyl-tRNA to the A site . Evidence from affinity-labeling studies with tRNA derivatives containing photoreactive nucleosides shows that L27 cross-links predominantly when bound to ribosomal A or P sites .

The proximity of L27 to the peptidyltransferase center is confirmed by cross-linking studies showing it must be within 2-4 Å of the azido group at the 3′ terminus of tRNA . This positions L27 (and by extension, RPL27A) close to the active site for peptide bond formation. This finding is particularly significant because it represents a potential difference between bacterial and archaeal ribosomes, as no proteins are seen within 20 Å of this site in archaeal ribosome crystal structures .

How do mutations in RPL27A affect ribosome function and cell viability?

While specific RPL27A mutations aren't detailed in the search results, the impact of ribosomal mutations can be inferred from comprehensive mutation studies of the ribosomal active site. Using the iSAT platform, researchers have characterized how single nucleotide changes in critical regions affect translation activity and ribosome assembly .

Mutations can significantly impact:

• Translation efficiency: Similar to the A2451C mutation which reduces protein synthesis activity 2-fold .

• Ribosome assembly: Mutations may lead to accumulation of precursor particles or altered subunit ratios .

• Polypeptide synthesis accuracy: Some mutations affect translation readthrough at premature stop codons, which can serve as a proxy for translation accuracy or release factor fidelity .

In cell viability studies, deletion of the related L27 in E. coli demonstrates that this protein is essential for normal cellular function . By extension, significant mutations in RPL27A likely impact cell growth and viability through compromised protein synthesis.

How conserved is RPL27A structure and function across species?

RPL27A shows remarkable conservation across diverse species. The Drosophila melanogaster RPL27A is homologous to rat and yeast RPL27A and to other members of the L15 ribosomal protein family . This conservation extends to both sequence and function, highlighting the fundamental importance of this protein in ribosomal assembly and protein synthesis.

Comparative analysis of related ribosomal proteins provides further evidence of functional conservation. For example, studies of L27 from the extreme thermophile Aquifex aeolicus and its homologue from Escherichia coli show that despite some structural differences, the A. aeolicus protein can be incorporated into E. coli ribosomes and partially rescue the growth defect of an L27 deletion mutant .

What structural differences exist between prokaryotic and eukaryotic RPL27A homologs?

Structural differences between prokaryotic and eukaryotic RPL27A homologs can be inferred from studies comparing related ribosomal proteins. Analysis of L27 proteins from different bacterial species by circular dichroism and proton nuclear magnetic resonance spectroscopy revealed significant structural variations . The A. aeolicus L27 readily adopts a stable structure in solution, whereas the E. coli L27 is unstructured under the same conditions .

These structural differences likely reflect adaptations to different cellular environments and ribosomal architectures. The positioning of L27 at the peptidyltransferase center in bacterial ribosomes represents a significant difference from archaeal ribosomes . Similar structural differences may exist between prokaryotic and eukaryotic RPL27A homologs, potentially impacting their role in ribosome assembly and function.

Can RPL27A homologs from different species functionally substitute for each other?

Cross-species complementation studies with related ribosomal proteins provide insights into the functional exchangeability of RPL27A homologs. When A. aeolicus L27 was expressed in an E. coli L27 deletion mutant, it increased the growth rate and was incorporated into E. coli ribosomes . This suggests that despite evolutionary divergence, the core functional properties of these ribosomal proteins are conserved.

How can RPL27A be used as a tool in ribosome engineering studies?

RPL27A's position in the ribosome makes it a valuable target for ribosome engineering approaches. Drawing from methodologies used for studying ribosomal components, researchers can use RPL27A modifications to:

• Create specialized ribosomes: By introducing mutations in RPL27A, researchers may design ribosomes with altered substrate specificity or translation properties.

• Develop cell-free translation systems: Modified RPL27A could be incorporated into platforms like iSAT to create specialized protein synthesis systems .

• Study translation regulation: RPL27A variants could help elucidate mechanisms of translation control in different cellular contexts.

The iSAT platform described in search result provides a powerful approach for testing RPL27A variants without wild-type ribosome contamination, enabling rapid mutant construction and testing in a cell-like environment.

What methodologies can resolve conflicts in RPL27A structural and functional data?

When faced with contradictory data about RPL27A structure or function, researchers should employ complementary approaches:

• Integrated structural biology: Combine crystallography, cryo-EM, and solution-based methods like NMR to resolve structural discrepancies.

• Functional assays at multiple levels: Assess both in vitro activity (ribosome assembly, translation efficiency) and in vivo effects (growth rates, protein synthesis rates).

• Cross-species comparison: Analyze RPL27A behavior across multiple organisms to identify conserved versus species-specific features.

• Computational modeling: Use molecular dynamics simulations to reconcile experimental data with predicted structures and interactions.

This approach is exemplified in research on L27, where conflicting data about its proximity to the peptidyltransferase center was addressed through complementary techniques including cross-linking studies and structural analysis .

How can mutational analysis be applied to study RPL27A function?

Comprehensive mutational analysis, as demonstrated for ribosomal active sites , provides a powerful approach to understanding RPL27A function. Researchers can:

This approach has been successfully applied to study ribosomal active sites, where 180 single point mutations were constructed and characterized for their effects on translation activity and ribosome assembly .