Recombinant Escherichia coli Ribosome-recycling factor (frr)
Description
Introduction
The ribosome-recycling factor (RRF), encoded by the frr gene in Escherichia coli, is a critical protein essential for the fourth step of bacterial translation: disassembling the post-termination complex (PTC) to recycle ribosomes for subsequent rounds of protein synthesis. This process ensures efficient translation by releasing ribosomes from mRNA and tRNA after termination codon recognition . Below is a detailed analysis of its structure, function, and genetic studies, supported by experimental data and structural insights.
Structure of RRF
The crystal structure of E. coli RRF (PDB ID: 1EK8) reveals an L-shaped molecule composed of two distinct domains:
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Domain A: A triple-stranded antiparallel coiled-coil resembling the anticodon arm of tRNA, capable of binding the ribosome’s A-site.
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Domain B: An alpha/beta domain that facilitates domain movement and binding to the ribosome .
This tRNA mimicry allows RRF to function as a "molecular mimic," enabling recognition and disassembly of the PTC .
Functional Role in Translation
RRF’s primary function is to dissociate the 70S ribosome from mRNA and tRNA after termination, recycling ribosomal subunits for new translation initiation . Studies using temperature-sensitive frr mutants demonstrate that RRF deficiency leads to:
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Accumulation of ribosomes in 3′-UTRs due to incomplete recycling .
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Re-initiation of translation downstream of termination codons, bypassing canonical initiation signals .
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Bactericidal effects during transitions between growth phases, underscoring RRF’s essentiality .
Table 1 summarizes key functional outcomes of RRF depletion:
Genetic and Mutational Studies
Mutational analysis of frr reveals critical domains and residues:
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Domain C (C-terminal helix) contains Arg132, a hotspot for null mutations (5/52 independent isolates) .
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Domain B (coil-helix structure) is enriched in null mutations, suggesting structural importance .
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Intergenic suppressors restore partial function in temperature-sensitive mutants, highlighting compensatory interactions with other translation factors .
Table 2 categorizes frr mutations:
| Mutation Type | Description | Frequency | Domain Affected |
|---|---|---|---|
| Null | Loss of RRF function | 52/63 | Domain B (52%) |
| Reversion | Partial restoration of function | 6/63 | Domain A/C |
| Silent | No functional impact | 5/63 | Outside domains |
Homologues and Cross-Species Analogs
A plant homologue, RRFHCP (ribosome recycling factor homologue in chloroplasts), shares 46% sequence identity with E. coli RRF but lacks functional equivalence in bacterial systems . Its bactericidal effect on E. coli suggests structural divergence despite sequence conservation .
Product Specs
Target Background
KEGG: ecy:ECSE_0171
Q&A
What is the structural organization of E. coli RRF?
The crystal structure of E. coli RRF reveals an L-shaped molecule consisting of two distinct domains: a triple-stranded antiparallel coiled-coil domain and an alpha/beta domain. The coil domain has a cylindrical shape with a negatively charged surface, reminiscent of the anticodon arm of tRNA and domain IV of elongation factor EF-G. This structure suggests that RRF binds to the ribosomal A-site through its coil domain, effectively mimicking tRNA . The relative positions of these two domains can change around a hydrophobic cleft in the hinge region, where detergent molecules have been observed to bind in crystal structures .
How does RRF function in the context of protein synthesis?
RRF works in concert with either elongation factor G (EF-G) or release factor 3 (RF3) to catalyze the disassembly of post-termination ribosomal complexes . After translation termination, RRF binds to the ribosome and, with the help of EF-G and GTP hydrolysis, facilitates the release of mRNA and deacylated tRNA, thereby recycling ribosomes for new rounds of translation . This recycling process is essential for efficient protein synthesis and bacterial survival, as evidenced by the fact that RRF is an essential protein in prokaryotes .
What molecular interactions underlie the species-specific functionality of RRF?
The species-specific interaction appears to involve the release of RRF from ribosomes by its cognate EF-G. In E. coli, ec-RRF is released from ribosomes by ec-EF-G through a mechanism where RRF is moved from its high-affinity A/P-site to a second lower-affinity site . Studies with tt-RRF show that while it can bind to E. coli ribosomes, it specifically requires tt-EF-G (not ec-EF-G) for its release from these ribosomes . This specificity highlights the precise molecular choreography required for ribosome recycling and provides insights into potential intermediate states during this process.
How do plant RRF homologues compare to bacterial RRF?
A protein designated as mature RRFHCP has been isolated from chloroplasts of spinach (Spinacia oleracea L.) that shows 46% sequence identity and 66% sequence homology with ribosome recycling factor of Escherichia coli . From cDNA analysis and amino-terminal sequencing, mature RRFHCP has a molecular weight of 21,838 with 193 amino acids, lacking the 78-amino acid chloroplast targeting sequence encoded by the RRFHCP cDNA sequence . This homology reflects the prokaryotic origin of chloroplast translation machinery and provides evidence for evolutionary conservation of the ribosome recycling mechanism.
Can plant RRF homologues functionally replace bacterial RRF?
Complementation assays demonstrate that mature RRFHCP cannot functionally replace E. coli RRF. When a plasmid carrying truncated frrhcp (coding for mature RRFHCP) was introduced into an E. coli strain with a nonfunctional chromosomal RRF gene, 100% of the transformants retained the resident plasmid carrying wild-type E. coli frr . This indicates an absolute requirement for wild-type E. coli frr, even in the presence of the plant frr homologue, demonstrating that t-RRFHCPfrr does not function in E. coli .
What effects do plant RRF homologues have on bacterial systems?
Intriguingly, mature RRFHCP exerts a bactericidal effect on E. coli carrying temperature-sensitive RRF at the permissive temperature, while having no effect on wild-type E. coli . This bactericidal action was demonstrated both during lag and logarithmic growth phases, with a decrease in colony-forming units upon expression of the plant gene . The selective inhibitory effect was confirmed by in vitro experiments showing that the activity of temperature-sensitive RRF was almost completely inhibited by a 2-fold excess of mature RRFHCP, whereas even a 30-fold excess inhibited wild-type RRF by only 70% . This phenomenon suggests that RRFHCP may interfere with the already compromised function of temperature-sensitive RRF.
What are the standard methods for measuring RRF activity?
RRF activity can be measured using an established assay where E. coli polysomes are isolated and treated with puromycin to remove nascent peptide chains . The conversion of these puromycin-treated polysomes to monosomes, catalyzed by EF-G and RRF, serves as the basis for determining RRF activity . This assay is typically conducted at 32°C and provides a quantitative measure of RRF function in vitro .
How can researchers assess functional complementation of RRF in vivo?
Complementation assays utilizing E. coli strains with defective chromosomal RRF genes provide a powerful approach for assessing the functionality of RRF variants. The table below illustrates a typical complementation experiment:
| E. coli strains | Incoming plasmid | Resident plasmid | Percentage of E. coli with frrhcp retaining resident plasmid A |
|---|---|---|---|
| a | pKK233-2RRFM | A | 100 |
| b | pKK233-2RRFM | A + B | 9.4 |
| a | pRR2 | A | 12.5 |
| b | pRR2 | A + B | 0 |
| a | pUC19 | A | 100 |
| b | pUC19 | A + B | 0 |
In this system, plasmid A carries wild-type E. coli frr and a kanamycin-resistance gene (Kmᵣ), while the incoming plasmid carries the test RRF gene and an ampicillin-resistance gene (Apᵣ) . The retention or loss of plasmid A (determined by kanamycin resistance) indicates whether the test RRF gene can functionally replace wild-type E. coli frr . For example, when mature RRFHCP (pKK233-2RRFM) was introduced, 100% of transformants retained plasmid A, indicating that RRFHCP cannot replace E. coli RRF .
What techniques are used to investigate bactericidal effects of RRF variants?
To study the bactericidal effects of RRF variants, researchers can transform E. coli strains with plasmids expressing the variant under an inducible promoter . For instance, E. coli LJ2221 (with temperature-sensitive RRF) harboring pKK233-2RRFM (carrying truncated frrhcp) can be exposed to isopropyl β-d-thiogalactoside to induce production of mature RRFHCP . Colony-forming units can then be measured over time to assess bactericidal effects . In vitro experiments comparing the inhibitory effects of RRF variants on wild-type versus temperature-sensitive RRF activity provide additional mechanistic insights .
What is the mechanism of RRF and EF-G interaction during ribosome recycling?
Current research suggests a model where RRF initially binds to the ribosomal A/P-site through its tRNA-mimicking domain . EF-G then catalyzes the movement of RRF from this high-affinity site to a second lower-affinity site, which likely overlaps with the E-site . The release of RRF from ribosomes is a vital part of the disassembly reaction, and the release of mRNA is coupled to or closely related to this process . This mechanism represents a coordinated process that efficiently recycles ribosomes for new rounds of translation.
What is the biological significance of RRF in bacterial physiology beyond translation?
RRF has been identified as a heat-shock protein, and bacterial cellular RRF concentration is elevated during the infection of animals by that bacteria . Furthermore, the amount of antibacterial RRF antibodies is high in some infected animals, suggesting that RRF may play an important role in bacterial pathogenesis . These observations point to broader physiological roles for RRF beyond its canonical function in ribosome recycling and highlight its potential importance in bacterial adaptation to stress conditions and host-pathogen interactions.
