Recombinant Putative RelE-like toxin protein

What are RelE-like toxins and how do they function?

RelE-like toxins are endoribonucleases belonging to the widespread type II ParE/RelE superfamily in bacterial genomes. They function by cleaving transcripts that are being translated, effectively shutting down translation in bacterial cells . Most RelE toxins operate in a translation-dependent manner and require the ribosome for proper RNA binding and cleavage . Unlike some ribonucleases, RelE has lost conserved histidine and glutamate residues typically used for RNA cleavage and instead relies heavily on conserved basic residues for both interaction and catalysis . The toxin leaves 2′-3′-cyclic phosphate at the new 3′ end after cleavage .

What distinguishes RelE toxins from other toxin families?

RelE toxins are part of the ParE/RelE superfamily but function distinctly from ParE toxins. While RelE toxins act as endoribonucleases, ParE family members inhibit DNA-gyrase and DNA replication . Despite functional divergence, they share common ancestry as evidenced by:

• Some conserved residues across the RelE/ParE superfamily

• Strikingly similar three-dimensional structures

• Conserved principle of binding to their cognate antitoxins

The major structural differences include extended N-terminal alpha helices and absence of C-terminal helix in ParE toxins, along with ParE lacking the major catalytic residues used by RelE for mRNA cleavage .

What is the cleavage specificity pattern of RelE toxins?

While RelE toxins exhibit specific cleavage patterns, these patterns vary between family members. Generally, RelE toxins tend to cleave:

• Upstream of purines

• Between the second and third positions of codons (except for actinobacterial toxins)

For the canonical RelE toxin, there is preferential cleavage at the second position of stop codons, with different rates observed among stop codons (UAG>UAA>UGA) . Some RelE-like toxins like the one from H. pylori preferentially cleave upstream of purines, while others such as the RelE-like toxin from Proteus vulgaris preferentially cleave at AAA sequences .

What expression systems are most effective for recombinant RelE-like toxins?

The expression of recombinant RelE-like toxins requires careful consideration due to their toxic nature. Effective expression systems include:

• Tightly regulated promoter systems: The use of arabinose-inducible (ara) promoters has been successful for expression of RelE toxins, allowing controlled induction of the toxic protein .

• Co-expression with cognate antitoxin: When studying the entire TA module, co-expression with the antitoxin neutralizes the toxic effects and enables higher expression yields .

• Affinity tag options: Both GST-tag and His-tag systems have proven effective for purification of RelE-like toxins .

For example, the novel Xn-relE toxin from Xenorhabdus nematophila was successfully expressed and purified under native conditions using GST and Ni-NTA chromatography, confirming the existence of this TA module .

How can researchers overcome the toxicity challenges when expressing RelE proteins?

Several strategies can mitigate toxicity issues during RecE protein expression:

• Use of tight regulatory control: Employ promoters with minimal leaky expression. The tightly regulated arabinose promoter system has proven effective for controlling RelE expression .

• Strategic mutations: Consider modifying the stop codon of the toxin gene. For instance, mutating the stop codon from UGA to UAA can make RelE toxin inhibit its own translation more moderately, as demonstrated in the BBa_K185047 construct .

• Co-expression approach: Express the toxin along with its cognate antitoxin to neutralize toxicity. This approach was successfully employed with Xn-relE toxin and its antitoxin Xn-relEAT .

• Optimized expression conditions: Lower temperature, reduced induction levels, and shorter induction times can help balance toxin production and cellular viability.

What methods are most effective for assessing RelE toxin activity?

Multiple complementary approaches can be used to analyze RelE toxin activity:

• Endogenous toxicity assays: Express the recombinant toxin under a tightly regulated promoter (like the ara promoter) in bacterial cells such as E. coli Top 10 and monitor growth inhibition. This can be complemented with neutralization assays using the cognate antitoxin to confirm specificity .

• Primer extension analysis: This technique has been effectively used to identify cleavage patterns of RelE toxins in vivo, revealing specific signature patterns in terms of frequency and location of cleavage sites .

• Translation inhibition assays: Measure the impact on protein synthesis rates in cell-free translation systems before and after toxin addition.

• Colony formation assays: RelE expression has been shown to prevent colony formation, making this a useful quantitative measure of toxin activity .

How do researchers analyze the RNA cleavage specificity of RelE toxins?

To analyze cleavage specificity, researchers can employ these techniques:

• Primer extension analysis: This approach identifies the exact cleavage sites on target mRNAs. For example, this method revealed that many RelE toxins tend to cleave upstream of purines and between the second and third positions of codons .

• Transcript-specific analysis: Testing cleavage patterns on specific transcripts such as lpp and ompA in E. coli has been effective in characterizing RelE activity .

• Codon preference analysis: For RelE toxins that show codon specificity, comparing cleavage rates at different stop codons (UAG, UAA, UGA) can reveal preferential activity .

• Ribosome dependency tests: Comparing cleavage activity with and without functional ribosomes helps distinguish between translation-dependent and translation-independent RelE-like toxins .

What experimental controls are essential when characterizing novel RelE-like toxins?

When characterizing novel RelE-like toxins, the following controls are essential:

• Antitoxin neutralization control: Expression of the cognate antitoxin should neutralize the toxin's effects, confirming the specificity of the observed toxic activity .

• Inactive mutant control: Expression of a catalytically inactive mutant of the RelE toxin can distinguish between specific toxic activity and general effects of protein overexpression.

• Translation dependency control: Comparing RNA cleavage with and without translation inhibitors (such as chloramphenicol) can verify whether the toxin requires active translation for function .

• Cross-species validation: Testing activity in different bacterial hosts can confirm conserved mechanism or reveal host-specific requirements.

How can RelE-like toxins be used as research tools in molecular biology?

RelE-like toxins offer several valuable applications as research tools:

• Regulated gene expression systems: The toxin-antitoxin module can be engineered as a molecular switch for controlling gene expression. For example, researchers have constructed UV-inducible systems using the sulA promoter and relE toxin to regulate bacterial growth in response to external stimuli .

• Ribosome interaction studies: Since many RelE toxins interact with the ribosome, they can serve as probes for studying ribosomal A-site structure and function.

• Translation termination research: RelE's preferential cleavage at stop codons makes it useful for investigating translation termination mechanisms and the role of release factors.

• Stress response models: RelE activation occurs under various stress conditions, making these toxins valuable for studying bacterial stress response pathways.

What evolutionary insights can be gained from studying RelE-like toxin diversity?

Studying the diversity of RelE-like toxins provides several evolutionary insights:

• Functional divergence: The RelE/ParE superfamily demonstrates how related proteins can evolve distinct functions - either as endoribonucleases (RelE family) or as inhibitors of DNA replication (ParE family) .

• Horizontal gene transfer: Toxin-antitoxin systems spread through horizontal gene transfer, which explains their presence across diverse bacterial species despite sequence divergence .

• Structural conservation despite sequence divergence: RelE-like sequences are quite divergent across species (often showing only 15-28% identity with canonical RelE K-12), yet they maintain similar predicted secondary and tertiary structures . This suggests strong selective pressure on structure rather than primary sequence.

• Adaptation to host-specific needs: The diverse cleavage specificities observed among RelE toxins may reflect adaptation to the specific translational machinery or stress response requirements of different bacterial hosts.

What are the challenges in distinguishing true RelE-like toxins from other related proteins?

Identifying authentic RelE-like toxins presents several challenges:

• Sequence divergence: RelE-like sequences can be highly divergent, with as little as 15-28% identity to canonical RelE proteins, making sequence-based identification difficult .

• Functional verification requirement: Bioinformatic prediction alone is insufficient; functional verification through expression and activity assays is necessary to confirm true RelE-like toxins.

• Overlap with ParE superfamily: The structural similarity between RelE and ParE toxins means that distinguishing between these functionally distinct families requires careful analysis of key catalytic residues and structural elements .

• Mixed mechanistic features: Some RelE-like toxins show characteristics of other RNases, creating a continuum rather than discrete categories. For example, while canonical RelE uses basic residues for catalysis, others like HigB, YoeB, and YafQ function more like RNase T1 through conserved histidine and glutamate residues .

What are common pitfalls in experimental design when studying RelE-like toxins?

Several experimental design issues can complicate RelE toxin research:

• Inadequate promoter control: Leaky expression from insufficiently regulated promoters can lead to growth inhibition before induction, selection of escape mutants, and poor reproducibility.

• Neglecting translation dependency: Some RelE toxins require active translation for proper function, so experimental conditions that inadvertently inhibit translation may give false negative results .

• Misattribution of activity: Without proper controls, general stress responses from protein overexpression might be mistaken for specific toxin activity.

• Species-specific effects: RelE homologs from different bacterial species may have different requirements for optimal activity, which should be considered when expressing them in heterologous hosts .

• Failure to create well-defined research questions: As with any research, inadequate formulation of research questions can lead to poorly designed experiments. Research questions should be logical, flowing from known facts to unknowns requiring validation .

How should researchers approach the formulation of research questions about RelE-like toxins?

When formulating research questions about RelE-like toxins, consider these guidelines:

• Ensure relevance: The question should be of academic and intellectual interest to people in the toxin-antitoxin field, preferably arising from issues in current literature or practice .

• Assess complexity: Research questions should not be answerable with a simple "yes" or "no" but should require both research and analysis .

• Evaluate feasibility: Consider whether the methodology to conduct the research is feasible within available time and resources .

• Structure question types:

  • Questions of description: These describe properties or characteristics of RelE toxins

  • Questions of relationship: These explore connections between RelE activity and other biological processes

  • Questions of comparison: These compare different RelE homologs or compare RelE to other toxin families

• Ensure measurability: The question should lead to a process that produces data that can be supported or contradicted .

What methodological considerations are important for retrospective analysis of RelE toxin research?

When conducting retrospective analyses of RelE toxin research, consider these methodological points:

• Clear research objectives: Formulate well-defined research questions that guide the study design and data analysis .

• Standardized data extraction: Develop a comprehensive data collection form that captures all relevant variables from primary studies, including experimental conditions, bacterial strains, and measurement methods.

• Quality assessment: Evaluate the methodological rigor of included studies, considering factors like appropriate controls, replication, and statistical analyses .

• Data heterogeneity: Account for variations in experimental approaches across different studies, which may impact the comparability of results.

• Publication bias awareness: Consider that published literature may overrepresent positive findings and underreport negative or inconclusive results.

• Avoid common retrospective review mistakes: Be aware of methodological pitfalls in retrospective reviews, such as poor definition of variables, inadequate sampling strategies, and failure to control for confounding factors .