Recombinant Anaerocellum thermophilum Ribosome-recycling factor (frr)

What is the function of ribosome-recycling factor in bacterial translation?

RRF, encoded by the frr gene, is responsible for dissociation of ribosomes from mRNA after the termination of translation. It functions to "recycle" ribosomes, making them available for new rounds of translation . Studies in E. coli have demonstrated that RRF is essential for bacterial growth, as evidenced by temperature-sensitive strains carrying frame-shifted frr in the chromosome . When RRF is depleted, post-termination 70S complexes accumulate in 3′-UTRs, and elongating ribosomes become blocked by non-recycled ribosomes at stop codons, preventing completion of translation .

How does RRF work mechanistically during ribosome recycling?

RRF works in concert with elongation factor G (EF-G) and GTP to split post-termination 70S ribosomes into 30S and 50S subunits. The process involves several steps: (1) binding of RRF to the A-site of post-termination complexes, (2) EF-G·GTP binding and GTP hydrolysis, which induces conformational changes, (3) dissociation of the deacylated tRNA, mRNA, and release factors, and (4) splitting of the 70S ribosome into subunits . This recycling process can be experimentally detected using sucrose density gradient ultracentrifugation, where the 70S peak decreases while 30S and 50S peaks increase following successful RRF activity .

What key domains and structural features characterize bacterial RRF proteins?

Bacterial RRF typically consists of two domains: a three-helix bundle domain that mimics tRNA structure, and a three-layered β/α/β sandwich domain connected by a hinge region. This architecture allows RRF to interact effectively with the ribosome and translation factors like EF-G. In thermophilic bacteria like A. thermophilum, the RRF protein likely possesses additional structural features that enhance thermostability, such as increased salt bridges, more compact hydrophobic cores, and reduced flexibility in loop regions .

How does RRF depletion impact global translation as revealed by ribosome profiling?

Ribosome profiling studies in E. coli have shown that RRF depletion leads to significant accumulation of ribosome footprints in 3′-UTRs, indicating the presence of post-termination 70S complexes that failed to be recycled . Additionally, elongating ribosomes form a queue behind stop codons, spaced one ribosome footprint apart, suggesting that post-termination complexes block elongation . Interestingly, contrary to previous hypotheses, RRF depletion did not significantly affect translational coupling efficiency within operons, indicating that re-initiation is not a major mechanism of translational coupling in bacteria .

What is the relationship between RRF and ribosome rescue factors?

RRF depletion has dramatic effects on the activity of ribosome rescue factors tmRNA and ArfA . After only 5 minutes of RRF depletion, there is a sharp increase in ribosome footprints on the short ORF within tmRNA, with the strongest peak at the stop codon . This suggests an accumulation of post-termination complexes that cannot be recycled. After 60 minutes of RRF depletion, ribosome footprints also accumulate downstream of the tmRNA ORF in regions that normally play important structural roles . These accumulated non-recycled ribosomes likely denature key structures in tmRNA, interfering with its ability to rescue stalled ribosomes and preventing degradation of ArfA at the mRNA and protein levels .

How might the thermostable properties of A. thermophilum RRF differ from mesophilic RRFs?

As a protein from a thermophilic organism growing optimally around 75°C, A. thermophilum RRF likely possesses specific adaptations that maintain its structure and function at elevated temperatures. These adaptations may include: (1) increased hydrophobic core packing, (2) higher content of charged amino acids forming extensive ion-pair networks, (3) reduced surface loop regions, and (4) increased proline content in loop regions. Structural studies comparing thermophilic and mesophilic RRFs would be needed to precisely identify the thermostabilizing features specific to A. thermophilum RRF .

What protocols are recommended for expression and purification of recombinant A. thermophilum RRF?

Expression of recombinant A. thermophilum RRF in E. coli typically involves:

• Cloning the frr gene into an expression vector with a suitable tag (e.g., His6)

• Transforming into an expression strain (e.g., BL21(DE3))

• Inducing expression with IPTG (typically at 30-37°C)

For purification, a recommended protocol based on similar proteins would include:

• Cell lysis in an appropriate buffer (e.g., 20 mM Tris-Cl, pH 7.5, 100 mM KCl)

• Heat treatment (65-75°C for 15-30 minutes) to denature E. coli proteins while leaving the thermostable A. thermophilum RRF intact

• Affinity chromatography (e.g., Ni-NTA for His-tagged proteins)

• Size exclusion chromatography using a Superdex 75 column

• Optional: additional purification via hydrophobic interaction chromatography

How can the activity of purified recombinant A. thermophilum RRF be assessed?

The activity of purified A. thermophilum RRF can be assessed using a ribosome splitting assay:

• Form reaction mixtures containing 0.2 μM 70S ribosomes, 20 μM RRF, 20 μM EF-G, 5 μM IF3, and 0.5 mM GTP in an appropriate buffer (e.g., Tris-polymix buffer with 6 mM Mg(OAc)₂)

• Incubate for 20 minutes at an appropriate temperature (37°C for standard assays, but higher temperatures may be used to test thermostability)

• Load reaction mixtures onto 10%-40% sucrose density gradients

• Perform ultracentrifugation (e.g., in an SW40 rotor at 25,000 rpm for 12 hours at 4°C)

• Analyze gradients by monitoring absorbance at 254 nm

• Active RRF will show decreased 70S peaks and increased 30S and 50S peaks compared to controls lacking RRF

What approaches can be used to study the thermostability of A. thermophilum RRF?

Several methods can be employed to characterize the thermostability of A. thermophilum RRF:

• Differential Scanning Calorimetry (DSC) to determine melting temperature (Tm)

• Circular Dichroism (CD) spectroscopy to monitor secondary structure changes at different temperatures

• Activity assays at various temperatures to determine the temperature optimum and stability range

• Limited proteolysis at different temperatures to identify regions of structural flexibility

• Comparative activity assays with mesophilic RRFs (e.g., from E. coli) to quantify thermostability differences

• X-ray crystallography or cryo-EM to determine the three-dimensional structure and identify stabilizing features

How can A. thermophilum RRF be applied in cell-free protein synthesis systems?

A. thermophilum RRF could significantly enhance thermostable cell-free protein synthesis systems:

• Incorporation into high-temperature cell-free translation systems (50-75°C) could improve recycling efficiency

• Pairing with other thermostable translation factors from A. thermophilum or other thermophiles would create more robust systems

• Such thermostable systems could be advantageous for expressing proteins that require higher temperatures for proper folding

• The enhanced stability could lead to longer-lasting cell-free reactions, improving protein yields

How might comparative studies between archaeal and bacterial ribosome recycling systems inform our understanding of A. thermophilum RRF?

While bacteria use RRF and EF-G for ribosome recycling, archaea employ the ABC-type twin-ATPase ABCE1 . Comparative studies could:

• Reveal evolutionary adaptations specific to extreme environments

• Identify common structural features required for ribosome binding across domains

• Uncover unique mechanisms for coupling nucleotide hydrolysis to ribosome splitting

• Provide insights into the development of thermostable recycling systems

What insights might cryo-EM studies of A. thermophilum RRF bound to ribosomes provide?

High-resolution cryo-EM studies of A. thermophilum RRF in complex with ribosomes could:

• Reveal specific interactions that enable function at high temperatures

• Identify conformational changes during the recycling process

• Provide structural insights into the coordination between RRF and EF-G

• Uncover any unique features of thermophilic ribosomes that facilitate recycling

• Inform the design of engineered RRF variants with enhanced properties