Recombinant Photobacterium profundum 50S ribosomal protein L20 (rplT)

What is the structural organization of P. profundum ribosomal protein L20?

The L20 protein consists of two distinctively defined domains: a linear N-terminal domain (NTD) and a globular C-terminal domain (CTD). The highly cationic NTD penetrates deeply into the 50S ribosomal subunit and interacts with helices 25, 40, and 41 of 23S rRNA. The CTD interacts with helices 40 and 41 of the 23S rRNA and with ribosomal proteins L21 and L31 at the surface of the ribosome . This structural arrangement facilitates L20's role as one of the initiating components of 50S ribosomal subunit synthesis.

What are the known functional domains of P. profundum L20 protein?

The functional domains of L20 have been identified through truncation studies. The NTD is primarily involved in ribosome assembly, as truncation mutations in this domain lead to the accumulation of precursors of the 50S ribosomal subunit . The CTD is responsible for binding to a pseudoknot structure and negatively regulates the expression of certain genes, including its own operon . This domain-specific functionality makes L20 important for both structural assembly of ribosomes and regulation of gene expression.

What are the optimal conditions for culturing P. profundum for rplT expression studies?

P. profundum should be cultured in 75% strength 2216 Marine Medium (28 g/l) at 15°C and 0.1 megapascal (MPa), unless different conditions are required for specific experiments . For high-pressure growth experiments, inoculation should be performed in heat-sealable plastic bulbs containing media with no gas space, then placed in pressure vessels . When conducting temperature-dependent studies, particularly examining cold-shock response, cultures can be shifted from optimal temperature to cold conditions (typically 20°C) to observe L20's role in adaptation .

What is the recommended protocol for cloning and expressing recombinant L20 from P. profundum?

Based on established protocols for P. profundum genes, recombinant L20 can be cloned using PCR amplification with the Expand Long Template PCR system (Roche Applied Science) . For introducing plasmids into P. profundum, tri-parental conjugations using the helper E. coli strain pRK2073 are recommended . For expression studies, it's important to note that antibiotics such as chloramphenicol (200 μg/ml) or streptomycin (150 μg/ml) may be used for selection in P. profundum . Construction of truncated variants, such as L20(ΔN) or L20(ΔC), can be valuable for domain-specific functional studies .

How does L20 contribute to 50S ribosomal subunit assembly in P. profundum?

L20 serves as one of the initiating components of 50S ribosomal subunit synthesis. Its NTD penetrates into the core of the 50S subunit, interacting with specific helices of 23S rRNA, while its CTD forms interactions at the ribosome surface . Truncation experiments have demonstrated that mutations in the NTD lead to accumulation of precursors of the 50S ribosomal subunit, confirming its direct role in ribosome assembly . The protein likely functions as a nucleation center, facilitating the correct folding and assembly of rRNA and other ribosomal proteins during 50S subunit biogenesis.

What methodologies are used to study the impact of L20 mutations on ribosome assembly?

Ribosome assembly can be analyzed using sucrose gradient density sedimentation to separate and quantify ribosomal subunits and polysomes . For L20 mutation studies, researchers typically:

• Generate truncated variants of L20 (such as L20(ΔN) and L20(ΔC))

• Express these variants in appropriate strains (e.g., the ESC19 strain with bipA deletion)

• Culture cells at both optimal (37°C) and cold-shock (20°C) temperatures

• Harvest cells and prepare lysates for sucrose gradient analysis

• Compare polysome profiles and subunit distribution between wild-type and mutant L20 expressions

This approach allows visualization of ribosome assembly defects resulting from specific L20 mutations .

How does L20 function change under cold-shock conditions in P. profundum?

Under cold-shock conditions (20°C), L20 appears to play a critical role in proper ribosome assembly. Research indicates that overexpression of L20 can rescue growth defects in strains lacking BipA (a cold-shock-inducible ribosome-associating GTPase) at low temperatures . This suggests that L20 has enhanced importance for ribosome biogenesis during cold stress. The suppressive effect of L20 on cold-sensitivity in bipA-deleted strains is primarily mediated through its N-terminal domain, as L20(ΔC) can still partially restore growth while L20(ΔN) shows greater cold-sensitivity .

What evidence supports the functional relationship between L20 and BipA in P. profundum?

Several lines of evidence support a functional relationship between L20 and BipA:

• Overexpression of rplT (encoding L20) restores normal growth of BipA-deleted strains under cold-shock conditions

• L20 partially recovers the defects in ribosomal RNA processing and ribosome assembly seen in BipA-deficient strains

• The suppressive effect appears to be mediated through L20's role in ribosome assembly rather than through translational regulation

This suggests that BipA and L20 may have coordinated actions in ensuring proper ribosome assembly, particularly under cold-shock conditions .

What experimental approaches can be used to investigate L20-BipA interactions in ribosome assembly?

To investigate L20-BipA interactions in ribosome assembly, researchers could employ:

• Co-immunoprecipitation studies: Using antibodies against L20 or BipA to pull down potential protein complexes

• Genetic suppressor screening: As demonstrated in the literature, where L20 was identified as a suppressor of BipA deletion

• Ribosome profiling: To identify the positioning of ribosomes on mRNAs in strains with various L20 and BipA mutations

• Cryo-electron microscopy: To visualize structural changes in ribosome assembly with and without L20 or BipA

• In vitro reconstitution assays: Using purified components to assess the direct effects of L20 and BipA on ribosome assembly

These approaches could help elucidate whether L20 and BipA interact directly or function in parallel pathways affecting ribosome assembly.

How can researchers differentiate between the structural and regulatory roles of L20 in P. profundum?

To differentiate between structural and regulatory roles of L20, researchers can:

• Generate domain-specific mutations (as shown with L20(ΔN) and L20(ΔC))

• Construct translational fusions (such as pRS414-P) to monitor regulatory effects

• Perform ribosome assembly assays to assess structural contributions

• Use RNA binding assays to characterize interactions with specific rRNA regions

• Employ complementation studies with heterologous L20 proteins from other species

The data showing that L20(ΔC) can restore growth better than L20(ΔN) in BipA-deleted strains suggests that the structural role in ribosome assembly (primarily mediated by the NTD) may be more critical than the regulatory function (primarily mediated by the CTD) under cold-shock conditions .

What methodological approaches can be used to study the rRNA binding properties of P. profundum L20?

To study rRNA binding properties of P. profundum L20, researchers can employ:

• Electrophoretic Mobility Shift Assays (EMSA): To detect protein-RNA interactions in vitro

• RNA footprinting: To identify specific nucleotides protected by L20 binding

• Surface Plasmon Resonance (SPR): To measure binding kinetics and affinity constants

• UV cross-linking: To capture transient RNA-protein interactions

• Structural studies: Using X-ray crystallography or cryo-EM to visualize L20-rRNA complexes

These techniques could help characterize the interactions between L20 domains and specific rRNA helices (25, 40, and 41 of 23S rRNA) that have been implicated in ribosome assembly .

How does P. profundum L20 compare with L20 proteins from other bacterial species?

While the provided search results don't directly address this comparison, research approaches could include:

• Sequence alignment analysis of L20 proteins from various bacterial species

• Structural comparison through homology modeling

• Functional complementation studies to determine if L20 from other species can replace P. profundum L20

• Examination of domain conservation, particularly between piezophilic and non-piezophilic bacteria

Such comparisons would be particularly valuable between L20 from piezophilic bacteria (like P. profundum SS9) and piezosensitive bacteria (like P. profundum 3TCK or E. coli) to identify potential pressure-adapted features.

Can P. profundum L20 functionally replace L20 in mesophilic or thermophilic bacteria?

This question could be addressed through heterologous expression studies where P. profundum L20 is expressed in L20-depleted strains of mesophilic (e.g., E. coli) or thermophilic bacteria. Key aspects to investigate would include:

• Growth complementation at various temperatures

• Ribosome assembly efficiency

• Translational fidelity and rates

• Stress response capabilities

Such studies could reveal temperature-specific adaptations in the structure and function of L20 across different bacterial species.