Recombinant Teredinibacter turnerae Peptide chain release factor 1 (prfA)
Product Specs
Target Background
KEGG: ttu:TERTU_3844
STRING: 377629.TERTU_3844
Q&A
Recombinant Teredinibacter turnerae Peptide Chain Release Factor 1 (prfA): Research FAQs
What is the functional role of prfA in Teredinibacter turnerae, and how does it intersect with translation termination mechanisms?
Basic Q: Peptide chain release factor 1 (prfA) in Teredinibacter turnerae is a protein critical for terminating translation during ribosomal protein synthesis. In bacteria, prfA (also known as RF1) recognizes stop codons (UAA and UAG) and facilitates the release of nascent polypeptides from ribosomes. Unlike eukaryotic systems, bacterial release factors lack a conserved domain for stop codon recognition, relying instead on a distinct mechanism involving conformational changes during translation.
Advanced Q:
Experimental validation of prfA’s role in T. turnerae requires cross-species functional complementation assays. For example, researchers could disrupt the prfA gene in T. turnerae and assess growth defects under conditions requiring robust translation termination. Additionally, structural studies (e.g., X-ray crystallography or cryo-EM) could elucidate how prfA interacts with ribosomal subunits and stop codons in this symbiont.
How might prfA expression levels correlate with environmental stressors in T. turnerae’s symbiotic lifestyle?
Basic Q: In symbiotic bacteria like T. turnerae, prfA expression may be regulated by environmental factors such as iron availability, cellulose abundance, or host-derived signals. Under iron limitation, T. turnerae upregulates siderophore biosynthesis (e.g., turnerbactin), which requires efficient translation of iron-scavenging proteins. prfA expression could rise under these conditions to optimize translation termination.
Advanced Q:
To investigate this, researchers could perform transcriptomic or proteomic profiling of T. turnerae under varying iron concentrations or cellulose-rich environments. Comparative analysis with mutants lacking prfA would reveal whether translation termination efficiency impacts siderophore production or cellulolytic enzyme synthesis.
What methodologies are best suited for studying prfA’s interactions with ribosomal complexes in T. turnerae?
Basic Q: Recombinant prfA production in heterologous systems (e.g., E. coli) allows for biochemical assays to test ribosome-binding affinity and stop codon recognition. Techniques like fluorescence-based assays (e.g., using ribosomes labeled with fluorescent tRNA analogs) can quantify prfA activity in vitro.
Advanced Q:
For in vivo studies, ribosome profiling (Ribo-seq) in T. turnerae could map ribosome occupancy across mRNAs, identifying translation termination sites influenced by prfA. Mutational analysis of conserved prfA motifs (e.g., GGQ loop) combined with cryo-EM structures of T. turnerae ribosomes would provide mechanistic insights into release factor function.
How does prfA’s evolutionary conservation in gammaproteobacteria inform its functional specificity in T. turnerae?
Advanced Q:
Phylogenetic analysis of prfA across shipworm symbionts (e.g., Teredinibacter spp.) versus free-living relatives could reveal lineage-specific adaptations. Functional studies comparing T. turnerae prfA with homologs from Vibrio spp. or E. coli would test whether symbiosis-driven selection pressures have altered its activity or substrate specificity.
What challenges arise when using recombinant prfA for structural or biochemical studies, and how can they be mitigated?
Basic Q: Recombinant prfA production may face challenges such as misfolding, aggregation, or low solubility due to the protein’s intrinsic properties or heterologous expression conditions.
Advanced Q:
To address these, researchers could employ:
| Challenge | Solution |
|---|---|
| Low solubility | Use solubility-enhancing tags (e.g., GST, MBP) or optimize expression conditions (e.g., lower temperature, co-expression with chaperones). |
| Misfolding | Perform refolding from inclusion bodies using ion-exchange chromatography or size-exclusion chromatography. |
| Poor ribosome binding | Use T. turnerae-derived ribosomes for in vitro assays to mimic native conditions. |
How might prfA interact with other translation factors (e.g., RF2, ribosome rescue systems) in T. turnerae?
Basic Q: prfA likely works synergistically with RF2 (prfB), which recognizes UGA stop codons, and ribosome rescue systems (e.g., tmRNA) to ensure efficient translation termination.
Advanced Q:
Co-expression studies of prfA with RF2 or tmRNA in T. turnerae could reveal functional redundancy or cooperative interactions. Fluorescence resonance energy transfer (FRET) assays might track prfA’s recruitment to ribosomes during readthrough or rescue events.
What computational tools can predict prfA’s structural dynamics and functional domains in T. turnerae?
Basic Q: Bioinformatics tools like Phyre2, I-TASSER, or AlphaFold can predict prfA’s tertiary structure and identify conserved motifs critical for ribosome interaction.
Advanced Q:
Molecular dynamics simulations of prfA bound to ribosomal models could predict conformational changes during stop codon recognition. Comparative genomics tools (e.g., BLAST, HHpred) can identify prfA homologs in related symbionts and infer functional divergence.
How does prfA’s regulation intersect with stress response pathways in T. turnerae’s iron-limited or cellulose-rich environments?
Basic Q: Under iron limitation, T. turnerae upregulates siderophore biosynthesis (e.g., turnerbactin) and TonB-dependent transporters. prfA expression might increase to support translation of iron-acquisition proteins.
Advanced Q:
Chromatin immunoprecipitation sequencing (ChIP-seq) could identify transcription factors (e.g., Fur) regulating prfA under iron stress. Metabolic flux analysis linking translation termination rates to siderophore production would quantify prfA’s impact on iron homeostasis.
What are the key considerations for designing prfA knockout mutants in T. turnerae for functional studies?
Basic Q: Knockout mutants must account for potential redundancy with RF2 or rescue systems. Selection markers (e.g., antibiotic resistance cassettes) should avoid disrupting adjacent genes.
Advanced Q:
A dual prfA/prfB knockout would test whether RF2 compensates for prfA loss. Phenotypic analysis (e.g., growth defects, protein production) under iron limitation or cellulose utilization would reveal prfA’s non-redundant roles.
How can prfA studies in T. turnerae inform broader understanding of translation termination in symbiotic bacteria?
Basic Q: Basic Q: Advanced Q: prfA studies in T. turnerae could reveal adaptations to symbiosis, such as optimized translation termination for nutrient acquisition (e.g., cellulose degradation, nitrogen fixation). Comparative studies with free-living relatives would highlight evolutionary pressures shaping translation machinery in symbionts.
