Recombinant Photobacterium profundum 50S ribosomal protein L25 (rplY)

What is the function of 50S ribosomal protein L25 in Photobacterium profundum?

L25 is a crucial component of the 50S ribosomal subunit in P. profundum. It specifically binds to 5S rRNA to form a stable complex, constituting a separate domain of the bacterial ribosome along with proteins L18 and L5 . Unlike in Escherichia coli where L25 is non-essential, in P. profundum this protein appears to be important for ribosomal assembly and function under pressure conditions. Experimental methods to study L25 function include:

• Transposon mutagenesis of the rplY gene, which has revealed pressure-sensitive phenotypes in P. profundum

• RNA-Seq analysis comparing wild-type and mutant strains to identify transcriptional changes

• Complementation analysis reintroducing the wild-type rplY to verify phenotype restoration

What structural features characterize the L25 protein and its interaction with 5S rRNA?

L25 specifically binds to a domain of 5S rRNA known as loop E . Key structural characteristics include:

• In E. coli, the L25 protein forms a stable complex with 5S rRNA

• The protein contains specific binding domains that recognize 5S rRNA structure

• The amino-terminal segment is required for ribosomal binding (as shown in studies of the homologous protein in yeast)

Experimental approaches to study these interactions include:

• X-ray crystallography of the L25-5S rRNA complex

• RNA footprinting to identify binding sites

• Site-directed mutagenesis of conserved residues

How does L25 contribute to ribosome assembly in deep-sea bacteria?

L25 forms part of a critical interaction network in ribosome assembly:

• It binds specifically to 5S rRNA, serving as one of three proteins (L25, L5, L18) that interact with 5S rRNA in eubacteria

• L25 has been shown to interact with L16 ribosomal protein

• In deep-sea bacteria, L25 appears to be important for ribosome stability under high pressure conditions

Studies in P. profundum have shown that genes for ribosome assembly and function (including L25) are important for both low-temperature and high-pressure growth . Methodological approaches include:

• Screening transposon insertion libraries for pressure and cold sensitivity

• Ribosome profiling under various pressure conditions

• Identification of pressure-specific mutations in rplY

What techniques are most effective for expressing and purifying recombinant P. profundum L25 protein?

Recombinant expression of P. profundum L25 can be achieved through several methodological approaches:

Expression Systems:

• E. coli-based expression using pET vector systems with T7 promoter

• Cold-adapted expression hosts for better folding of psychrophilic proteins

• Codon optimization for the expression host

Purification Protocol:

• Affinity chromatography using His-tag or MBP fusion strategies

• Ion exchange chromatography as a secondary purification step

• Size exclusion chromatography for final polishing

Expression Optimization Parameters:

Protein solubility can be improved by using fusion partners like maltose-binding protein (MBP), as demonstrated in studies with ribosomal protein L25 .

How does high hydrostatic pressure affect the expression and function of rplY in P. profundum?

P. profundum is a piezophilic bacterium adapted to high pressure environments. Research indicates:

• Transcriptome analysis reveals that P. profundum is under greater stress at atmospheric pressure than at elevated pressure, reflecting its deep-sea origin

• The rplY gene expression may be regulated in response to pressure changes, though specific pressure-responsive elements in its promoter region have not been fully characterized

• In other deep-sea bacteria, ribosomal proteins show enhanced transcription when pressure is increased from 0.1 to 10 MPa

Experimental approaches to study pressure effects include:

• RNA-Seq comparison of gene expression at different pressures (e.g., 0.1 MPa vs. 28 MPa)

• Pressure-regulated reporter gene constructs

• Pressure-sensitive mutant isolation and characterization

• Complementation analysis under various pressure conditions

What role does L25 play in pressure and cold adaptation in deep-sea bacteria?

L25 appears to be part of the adaptation strategy for deep-sea bacteria:

• Genes for ribosome assembly and function, including L25, are important for both low-temperature and high-pressure growth in P. profundum

• Transposon mutagenesis has identified that DEAD box RNA helicases (important for ribosome assembly) are critical for both cold and pressure adaptation

In comparative studies:

• The genome of P. profundum SS9 codes for at least nine DEAD box helicases, almost twice as many as the genome of E. coli

• Similar expansion of DEAD box helicase genes has been observed in other psychrophilic bacteria, suggesting importance for temperature adaptation

Methodological approaches include:

• Comparative genomics across piezophilic and mesophilic bacteria

• Mutational analysis of L25 and associated proteins

• In vitro translation assays under varying pressure conditions

How can site-directed mutagenesis of rplY be used to study pressure adaptation mechanisms?

Site-directed mutagenesis of rplY provides insights into pressure adaptation:

Experimental Approach:

• Identify conserved residues by sequence alignment of L25 from piezophilic vs mesophilic bacteria

• Create point mutations in conserved regions using PCR-based mutagenesis

• Express mutant proteins and test for:

  • 5S rRNA binding capability

  • Pressure tolerance

  • Cold adaptation

Key Target Regions:

• N-terminal segment required for L25 binding to ribosomes

• Residues involved in 5S rRNA interaction

• Regions showing evolutionary divergence in deep-sea bacteria

Complementation analysis of mutants at different pressures (0.1 MPa vs. 28 MPa) can reveal which residues are critical for pressure adaptation .

What are the molecular mechanisms by which L25 contributes to ribosome stability under high pressure?

High pressure affects ribosome stability through several mechanisms:

• In vitro studies show that uncharged ribosomes dissociate at 60 MPa, while ribosomal proteins associated with mRNA and tRNA show improved stability up to 100 MPa

• Correlation between loss of cell viability and ribosome integrity at high pressure has been postulated

• Enhanced transcription of 30S and 50S ribosomal proteins has been observed when pressure increases from 0.1 to 10 MPa

L25's specific contributions likely include:

• Stabilizing 5S rRNA structure under pressure

• Maintaining critical interactions with other ribosomal proteins

• Potentially altered binding kinetics optimized for high-pressure environments

Research methodologies include:

• Differential scanning calorimetry of ribosomes under pressure

• Molecular dynamics simulations of L25-rRNA interactions

• FRET-based assays to measure binding affinities at different pressures

How does the structure and function of P. profundum L25 compare with homologs from non-piezophilic bacteria?

Comparative analysis reveals important differences:

Structural Comparisons:

P. profundum has a record number of ribosomal operons (16) with high intragenomic variability within the operons (4% nucleotide divergence) . Studies have shown that the proportion of ribotypes with longer stems is directly correlated to the optimal growth pressure in different P. profundum strains (r² = 0.97) .

Methodological approaches include:

• Homology modeling and molecular dynamics simulations

• Heterologous expression of L25 from different species in P. profundum rplY mutants

• Circular dichroism spectroscopy under various pressure conditions

What interactions occur between L25 and other molecular chaperones in deep-sea bacteria?

Research indicates potential interactions between L25 and molecular chaperones:

• In related systems, nascent chain-associated complex (NAC) and signal recognition particle (SRP) share the ribosomal protein L25 as a docking site

• SRP54 specifically interacts with L25, and this interaction may be important for protein targeting

• Purified NAC (which interacts with L25) can prevent protein aggregation in vitro, showing molecular chaperone properties

These interactions may be particularly important under pressure stress conditions, where proper protein folding becomes more challenging. Investigation methods include:

• Pull-down assays to identify L25 binding partners

• Yeast two-hybrid screening for protein-protein interactions

• Cryo-EM to visualize ribosome-associated factors

What emerging technologies can enhance our understanding of L25 function in P. profundum?

Several cutting-edge approaches show promise:

CryoEM Techniques:

• High-resolution imaging of P. profundum ribosomes under various pressure conditions

• Visualization of L25-5S rRNA interactions in native state

CRISPR-Cas9 Applications:

• Precise genome editing to create point mutations in rplY

• CRISPRi for conditional knockdown of expression

Ribosome Profiling:

• Detailed mapping of translation dynamics under pressure

• Identification of L25-dependent translation effects

In situ Structural Biology:

• Development of high-pressure NMR and X-ray crystallography

• Real-time monitoring of pressure effects on ribosome assembly

These technologies can provide unprecedented insights into the molecular mechanisms of pressure adaptation in deep-sea bacteria.