Recombinant Microcystis aeruginosa Ribosome-recycling factor (frr)

What is the primary function of ribosome recycling factor in bacteria?

Ribosome recycling factor (RRF) is responsible for the dissociation of ribosomes from mRNA after the termination of translation. It works in conjunction with elongation factor G (EF-G) to disassemble the post-termination ribosomal complex, effectively "recycling" ribosomes for subsequent rounds of protein synthesis . This recycling process is essential for efficient translation, as it prevents ribosomes from remaining bound to mRNA after reaching the termination codon . The ribosome recycling step requires GTP hydrolysis and involves the release of deacylated tRNA and mRNA from the ribosome .

Is the frr gene essential for bacterial growth?

Yes, the frr gene has been conclusively demonstrated to be essential for bacterial cell growth. This was established through experiments with Escherichia coli strain MC1061-2, which carried a frame-shifted frr in the chromosome and wild-type frr on a temperature-sensitive plasmid . This strain exhibited temperature-sensitive growth and could not segregate its frr-carrying plasmid under plasmid incompatibility pressure . All thermoresistant colonies that formed spontaneously had recovered wild-type frr function, either through re-exchange in the bacterial chromosome or through mutations that made the plasmid temperature-resistant . These observations definitively established frr as an essential gene for cell growth.

What is the structural relationship between RRF and tRNA?

The crystal structure of RRF reveals that it is a near-perfect mimic of tRNA. RRF has two domains (I and II) that correspond to the anticodon and acceptor arms of tRNA, respectively . This structural similarity is not merely coincidental but functional, as it allows RRF to interact with the ribosome in a manner similar to tRNA . The structural mimicry suggests that RRF might be translocated like tRNA on the ribosome during the disassembly of the post-termination complex .

How is the frr gene organized in bacterial genomes?

The frr gene exists in a highly conserved genomic arrangement across many bacterial species. In bacteria such as E. coli, Pseudomonas aeruginosa, and Bacillus subtilis, the arrangement follows the pattern rpsB-tsf-pyrH-frr, where rpsB encodes ribosomal protein S2, tsf encodes elongation factor Ts, and pyrH encodes UMP kinase . This conservation extends even to cyanobacteria, where the pyrH-frr arrangement is maintained . The consistent gene organization across diverse bacterial species suggests functional importance and co-evolution of these components of the translation machinery.

Table 1: Gene organization around frr in different bacterial species

What is the evolutionary significance of RRF in eukaryotes?

Although RRF homologues have been identified in eukaryotic cells, phylogenetic analysis suggests they were originally present within the prokaryotic RRF phylogenetic tree . This finding indicates that the ribosome recycling step catalyzed by RRF is specific to prokaryotic cells, and eukaryotic RRF is likely required for protein synthesis in organelles that are believed to have originated from prokaryotes, specifically mitochondria and chloroplasts . This evolutionary relationship supports the endosymbiotic theory of organelle origin and highlights the conservation of essential translation mechanisms across evolutionary time.

How can researchers purify recombinant RRF for functional studies?

Purification of recombinant RRF typically involves expression in a suitable host system (often E. coli) followed by chromatographic purification. Based on established protocols, researchers should consider the following methodology:

First, clone the frr gene into an expression vector with an appropriate tag (histidine tags are commonly used). Express the protein in E. coli under optimized conditions (temperature, IPTG concentration, and duration of induction) . Lyse the cells under native conditions and purify the protein using affinity chromatography. For studying binding interactions, additional purification steps such as ion-exchange or size-exclusion chromatography may be necessary to achieve high purity .

For functional verification, the purified RRF should be tested for its ability to release monosomes from polysomes in the presence of EF-G and GTP, using either homologous or heterologous ribosome systems .

What methods can be used to study RRF-ribosome interactions?

Several methodological approaches can be employed to study RRF-ribosome interactions:

• Binding assays: Incubate purified RRF with ribosomes in buffer containing appropriate ions (Mg²⁺, K⁺) and use micro-spin column filtration followed by western blotting to detect and quantify ribosome-bound RRF . The dissociation constant of RRF for the 50S subunit has been determined to be approximately 2 × 10⁻⁶ M using such methods .

• Functional assays: Follow the release of tRNA and mRNA from post-termination complexes using techniques such as sucrose gradient centrifugation . The conversion of polysomes to monosomes can be monitored to assess RRF activity .

• Structural studies: X-ray crystallography of RRF-ribosome complexes provides detailed information about binding sites and conformational changes. The crystal structure of a posttermination T. thermophilus 70S ribosome complexed with EF-G, RRF, and tRNAs has been solved at 3.5 Å resolution, revealing critical insights into the mechanism of ribosome recycling .

• Inhibitor studies: Various inhibitors of protein synthesis can be used to probe the mechanism of RRF action, as they have differential effects on tRNA and mRNA release .

How can heterologous RRF activity be assessed in ribosome recycling machinery?

Heterologous RRF activity can be assessed using purified recombinant RRF and polysome assays. The methodology involves:

• Preparing polysomes from a model organism (typically E. coli) by isolating ribosomes engaged in translation .

• Setting up a reaction mixture containing the purified recombinant RRF from the species of interest (e.g., Microcystis aeruginosa), along with purified EF-G from the same or different species, GTP, and appropriate buffer conditions .

• Incubating the reaction mixture and then analyzing the conversion of polysomes to monosomes using techniques such as sucrose gradient ultracentrifugation followed by UV absorbance monitoring at 260 nm .

This approach has successfully demonstrated that P. aeruginosa RRF is active in E. coli ribosome recycling machinery, representing the first case of an RRF homologue shown to be functional in a heterogeneous system . This suggests that the fundamental mechanism of ribosome recycling is conserved across bacterial species and that recombinant RRF from cyanobacteria such as Microcystis aeruginosa may similarly function in heterologous systems.

What is the detailed mechanism of ribosome recycling by RRF and EF-G?

The mechanism of ribosome recycling involves several coordinated steps:

• RRF binding: RRF binds to the A-site of the post-termination complex, which consists of the ribosome, deacylated tRNA in the P-site, and mRNA .

• Translocation-like movement: EF-G with GTP binds to the complex and catalyzes a translocation-like movement of RRF from the A-site to the P-site, similar to tRNA movement during translocation .

• tRNA release: This translocation results in the release of deacylated tRNA from the P- and E-sites of the ribosome .

• mRNA release: Following tRNA release, mRNA is released from the ribosome, accompanied by the release of RRF and EF-G .

• Ribosome dissociation: In the final step, the 70S ribosome released from mRNA can be dissociated into subunits by initiation factor 3 (IF3), preparing them for a new round of translation .

Experimental evidence supporting this mechanism includes the observation that inhibitors of translocation (thiostrepton, viomycin, aminoglycosides) inhibit both tRNA and mRNA release, while fusidic acid and GTP analogs (which allow one round of translocation but fix EF-G to the ribosome) inhibit mRNA release but not tRNA release .

How do inhibitors of protein synthesis affect RRF function?

Various inhibitors have different effects on RRF-mediated ribosome recycling, providing insights into the mechanism:

Table 2: Effects of inhibitors on RRF-mediated ribosome recycling

These differential effects support the model that RRF is translocated like tRNA on the ribosome during the recycling process . The release of tRNA is a prerequisite for mRNA release and partially occurs with EF-G alone, but complete ribosome recycling requires both RRF and EF-G working in concert .

What is the role of GTP hydrolysis in RRF-mediated ribosome recycling?

GTP hydrolysis is essential for RRF-mediated ribosome recycling . EF-G binds GTP and interacts with the ribosome-RRF complex, catalyzing a translocation-like movement of RRF from the A-site to the P-site . The energy derived from GTP hydrolysis drives conformational changes in EF-G and the ribosome that are necessary for the disassembly of the post-termination complex . This GTP-dependent process is similar to the role of GTP hydrolysis during the elongation phase of translation, reflecting the conservation of mechanistic principles in different phases of protein synthesis.

Why is RRF a potential target for antimicrobial development?

RRF represents an attractive target for antimicrobial development for several key reasons:

• Essentiality: The frr gene is essential for bacterial growth and viability, as demonstrated in E. coli studies . Inhibition of RRF function would therefore be lethal to bacteria.

• Specificity: The ribosome recycling step catalyzed by RRF is specific for prokaryotic cells . Although RRF homologues exist in eukaryotes, they function primarily in organelles rather than in cytoplasmic translation, potentially allowing for selective targeting of bacterial protein synthesis.

• Structural uniqueness: The detailed structural information available for RRF-ribosome complexes provides a foundation for structure-based drug design to develop inhibitors that specifically target bacterial RRF without affecting eukaryotic translation.

• Conservation: RRF is highly conserved across bacterial species, suggesting that inhibitors might have broad-spectrum activity against multiple bacterial pathogens.

What research gaps remain in our understanding of cyanobacterial RRF function?

Despite advances in understanding RRF function in model bacteria like E. coli, several knowledge gaps remain regarding cyanobacterial RRF, particularly from Microcystis aeruginosa:

How might studying the deacylated tRNA in the p/R state contribute to our understanding of ribosome dynamics?

Recent structural studies have revealed a previously uncharacterized state of deacylated tRNA binding (peptidyl/recycling, p/R) that is analogous to that seen during initiation . In this state, the terminal end of the p/R-tRNA forms non-favorable contacts with the 50S subunit while RRF wedges next to central inter-subunit bridges .

This discovery provides several important insights:

• It reveals a missing snapshot of tRNA as it transits between the P and E sites, filling a gap in our understanding of tRNA movement through the ribosome .

• It illuminates the active roles of both tRNA and RRF in the dissociation of ribosomal subunits, suggesting a more complex and coordinated process than previously thought .

• It provides a structural basis for understanding how RRF and tRNA cooperate to destabilize ribosomal subunit interactions, ultimately leading to their separation .

• It suggests mechanistic parallels between initiation and recycling phases of translation, potentially revealing conserved principles in ribosome function across different stages of protein synthesis.

Future research focusing on this p/R state could provide deeper insights into the energetics and dynamics of ribosome recycling and potentially reveal new targets for antimicrobial development.