Recombinant Clostridium kluyveri Ribosome-recycling factor (frr)

What is the function of Ribosome-recycling factor (frr) in Clostridium kluyveri?

Ribosome-recycling factor, encoded by the frr gene in Clostridium kluyveri, is responsible for the dissociation of ribosomes from mRNA after translation termination. It effectively "recycles" ribosomes, making them available for subsequent rounds of translation. Studies in E. coli have established that frr is an essential gene for cell growth, and this function is likely conserved in C. kluyveri . The protein functions in conjunction with elongation factors to ensure efficient protein synthesis by preventing ribosomes from stalling on mRNA after translation completion. Without proper ribosome recycling, cellular growth is severely compromised, highlighting the critical nature of this process in bacterial metabolism .

How does the genomic context of frr in Clostridium kluyveri compare to other bacterial species?

Clostridium kluyveri contains one circular chromosome of 3.96 Mbp and one circular 59-kb plasmid . The genome has several unique features compared to other clostridial genomes. The terminus of replication lies at approximately 150° of the chromosomal ring, with counterclockwise replication covering 210° of the chromosome, which is more extensive than in other sequenced clostridial genomes . The C. kluyveri genome exhibits a strong coding bias, with 76% of coding sequences encoded on the leading strand, similar to other clostridial and Bacillus genomes . While the specific genomic neighborhood of the frr gene isn't detailed in the available data, comparative genomic analysis would likely reveal its position relative to other translation-related genes, which is often conserved across bacterial species.

What experimental approaches are used to study the function of recombinant Clostridium kluyveri RRF in vitro?

Several sophisticated experimental approaches can be employed to investigate C. kluyveri RRF function:

• Expression and Purification Systems:

  • Recombinant expression in yeast systems with His-tag purification strategies

  • Recommended reconstitution in deionized sterile water to a concentration of 0.1-1.0 mg/mL with 5-50% glycerol for storage stability

• Functional Assays:

  • Ribosome dissociation assays measuring RRF's ability to release ribosomes from model mRNAs

  • Coupled GTPase activity assays when studying RRF's cooperation with elongation factors

  • Polysome profile analysis to assess the impact of RRF on ribosome distribution patterns

• Structural Biology Approaches:

  • X-ray crystallography or cryo-electron microscopy to determine RRF's interaction with ribosomal components

  • Site-directed mutagenesis to identify critical residues for function

• In vivo Complementation Studies:

  • Temperature-sensitive strain construction similar to the E. coli MC1061-2 model with frame-shifted frr in the chromosome and wild-type frr on a temperature-sensitive plasmid

  • Plasmid segregation assays under incompatibility pressure to assess gene essentiality

These methodologies provide comprehensive insights into both structural and functional aspects of RRF activity in the translation termination and ribosome recycling processes.

How do mutations in Clostridium kluyveri frr affect translation and cell viability?

While specific mutation studies in C. kluyveri frr aren't detailed in the available literature, insights can be drawn from E. coli studies:

• Essential Nature:

  • The frr gene is essential for cell viability as demonstrated in E. coli, where frame-shifted mutations resulted in temperature-sensitive growth

  • Cells carrying mutated frr require complementation with wild-type frr for survival

• Potential Effects of Mutations:

  • Reduced efficiency of ribosome recycling leads to ribosome sequestration and limited availability for new translation initiation

  • Accumulation of post-termination complexes (post-TCs) at stop codons

  • Ribosome profiling studies in other organisms show that depletion of recycling factors results in stacked ribosomes upstream of stop codons

  • Potential unintended re-initiation events in 3'-UTRs when recycling is impaired

• Compensatory Mechanisms:

  • Upregulation of alternative ribosome rescue factors (like ArfA in E. coli)

  • Increased expression of trans-translation systems involving tmRNA

These findings suggest that mutations in C. kluyveri frr would likely have profound effects on translation efficiency and cellular viability through disruption of ribosome availability and recycling processes.

What is the role of Clostridium kluyveri RRF in translational coupling and how can it be experimentally measured?

Translational coupling refers to the interdependent translation of adjacent genes in polycistronic mRNAs. Research on RRF's role in this process has yielded interesting insights:

• Current Understanding:

  • Contrary to earlier hypotheses, RRF depletion does not significantly alter the ratio of ribosome density on neighboring genes in polycistronic transcripts in E. coli

  • RRF depletion does not significantly change coupling efficiency in reporter assays of E. coli genes previously demonstrated to be translationally coupled

  • These findings suggest that re-initiation by ribosomes after recycling is not a widespread mechanism of translational initiation in bacteria

• Experimental Measurement Approaches:

  • Ribosome Profiling (Ribo-seq):

    • Quantitative analysis of ribosome-protected mRNA fragments across the genome

    • Measurement of ribosome density on adjacent genes in operons

    • Detection of ribosome accumulation at stop codons and in intercistronic regions

  • Reporter Systems:

    • Construction of bicistronic reporter constructs with varying intercistronic sequences

    • Measurement of downstream gene expression relative to upstream gene expression

    • Comparison of coupling efficiency between wild-type and RRF-depleted conditions

• Data Analysis Parameters:

  • Coupling efficiency calculations based on downstream:upstream expression ratios

  • Measurement of ribosome accumulation at stop codons as indicated by footprint density

  • Analysis of translation re-initiation events in untranslated regions

These approaches would provide valuable insights into whether C. kluyveri RRF plays a similar role in translational coupling as observed in E. coli or if species-specific differences exist.

Methodological Questions

Studying C. kluyveri RRF under anaerobic conditions requires specialized approaches that account for both the oxygen sensitivity of the organism and the global effects of RRF on translation:

• Anaerobic Experimental Setup:

  • Establish strict anaerobic culture conditions using specialized chambers with controlled atmosphere

  • Employ pre-reduced media suitable for C. kluyveri growth

  • Monitor redox potential to ensure maintenance of anaerobic conditions throughout experiments

• RRF Modulation Strategies:

  • Develop conditional expression systems for frr (temperature-sensitive mutants similar to the E. coli MC1061-2 model)

  • Construct strains with titratable frr expression using inducible promoters

  • Implement genetic approaches that enable controlled depletion of RRF protein

• Global Translation Analysis Methods:

  • Ribosome Profiling Under Anaerobic Conditions:

    • Harvest cells with minimal exposure to oxygen

    • Process samples rapidly to preserve authentic ribosome positions

    • Analyze ribosome footprint distribution across the transcriptome

    • Quantify ribosome accumulation at stop codons and in 3'-UTRs as indicators of recycling defects

  • Polysome Analysis:

    • Fractionate cell lysates on sucrose gradients under anaerobic conditions

    • Measure polysome:monosome ratios as indicators of translation efficiency

    • Compare profiles between wild-type and RRF-depleted conditions

• Specific Phenomena to Monitor:

  • Changes in tmRNA utilization and ribosome rescue pathways

  • Upregulation of alternative ribosome rescue factors

  • Effects on translation of specific gene classes (highly expressed genes, essential genes)

  • Impact on growth rate and metabolic activities specific to C. kluyveri's anaerobic metabolism

These approaches would provide comprehensive insights into how RRF functions within the context of C. kluyveri's unique anaerobic lifestyle and metabolism.

What bioinformatic approaches can be used to analyze the evolutionary conservation of Clostridium kluyveri RRF across bacterial species?

Several sophisticated bioinformatic approaches can reveal evolutionary patterns in C. kluyveri RRF:

• Sequence-Based Analyses:

  • Multiple sequence alignment of RRF proteins from diverse bacterial species

  • Calculation of sequence conservation metrics across taxonomic groups

  • Identification of universally conserved residues versus clade-specific variations

  • Construction of sequence logos to visualize conservation patterns at each position

• Phylogenetic Methods:

  • Maximum likelihood or Bayesian phylogenetic reconstruction of RRF evolution

  • Comparison of RRF phylogeny with species phylogeny to detect horizontal gene transfer events

  • Analysis of evolutionary rates to identify positions under purifying or diversifying selection

• Structural Bioinformatics:

  • Homology modeling of C. kluyveri RRF based on available crystal structures

  • Structural alignment with RRFs from diverse bacterial species

  • Mapping of sequence conservation onto three-dimensional structure

  • Identification of structurally conserved motifs essential for function

• Comparative Genomics:

  • Analysis of frr gene synteny across bacterial genomes

  • Identification of conserved gene neighborhoods that might indicate functional relationships

  • Comparison of regulatory elements in frr promoter regions across clostridial species

• Coevolution Analysis:

  • Detection of coevolving residues within RRF using statistical coupling analysis

  • Identification of coevolutionary relationships between RRF and its interaction partners

  • Inference of functional constraints from coevolutionary signatures

These bioinformatic approaches would provide valuable insights into the evolutionary history of RRF in Clostridium kluyveri and its functional conservation across the bacterial domain.

How does Clostridium kluyveri RRF differ functionally from RRF proteins in other bacterial species?

Comparative analysis of RRF across bacterial species reveals both conservation of core function and potential species-specific adaptations:

• Sequence and Structural Comparison:

  • C. kluyveri RRF consists of 185 amino acids, similar in length to RRFs from other bacterial species including Pseudomonas aeruginosa (185 aa), Bacillus subtilis (185 aa), and others listed in commercial catalogs

  • While core functional domains are conserved, sequence variations likely reflect adaptation to species-specific ribosomal components

• Functional Conservation:

  • The essential role of RRF in ribosome recycling appears universally conserved across bacterial species

  • E. coli studies demonstrate that RRF is essential for cell growth, a feature likely shared by C. kluyveri RRF

  • RRF works in concert with elongation factors in all bacterial species studied

• Species-Specific Differences:

  • Variations in sequence may reflect adaptations to different cellular environments

  • Interaction strength with partner molecules (EF-G, ribosomes) may vary between species

  • The impact on translational coupling appears less significant than previously hypothesized, at least in E. coli

• Ribosome Rescue Mechanisms:

  • RRF depletion in E. coli leads to upregulation of alternative ribosome rescue factors like ArfA

  • Species-specific differences in backup mechanisms for ribosome recycling may exist

  • The relative importance of RRF versus other rescue pathways may vary between species

Understanding these differences requires comparative biochemical studies using purified components from multiple species and cross-species complementation experiments.

What is the relationship between RRF function and the unique metabolic capabilities of Clostridium kluyveri?

Clostridium kluyveri possesses distinctive metabolic capabilities that may interact with translation and ribosome recycling in interesting ways:

• Metabolic Context:

  • C. kluyveri is a strict anaerobe with unique metabolic features, including the ability to form caproic acid and hexanol from ethanol and butyrate

  • The genome contains genes for nonribosomal synthesis of peptide-polyketide hybrids and enzymes for ethanol and glycerol fermentation to 1,3-propanediol

  • C. kluyveri possesses an extremely active sulfur metabolism with genes for sulfate adenylyltransferase, adenylylsulfate reductase, and other sulfur-processing enzymes

• Protein Synthesis Requirements:

  • C. kluyveri contains macromolecular complexes like ethanol dehydrogenase and acetaldehyde dehydrogenase that function in microcompartments similar to carboxysomes

  • Efficient translation and ribosome recycling would be essential for synthesizing these complex enzymatic systems

  • The genome contains multiple copies of certain metabolic genes, suggesting differential regulation of expression

• Translational Adaptation:

  • RRF function may be optimized for the particular demands of C. kluyveri's proteome

  • The efficiency of ribosome recycling could influence the expression of genes involved in unique metabolic pathways

  • Translation termination and recycling might be regulated differently under varying metabolic conditions

• Experimental Approaches:

  • Analysis of RRF activity under different metabolic conditions

  • Investigation of translational efficiency for key metabolic enzymes with and without RRF depletion

  • Assessment of how ribosome allocation changes during metabolic shifts in C. kluyveri

This relationship between RRF function and C. kluyveri metabolism represents an interesting intersection of translation efficiency and metabolic specialization that merits further investigation.