Recombinant Escherichia coli Peptide chain release factor 1 (prfA)
Description
Biological Role of prfA in Translation Termination
The prfA gene encodes Release Factor 1 (RF1), a protein responsible for recognizing the UAG and UAA stop codons during translation in E. coli. RF1 facilitates hydrolytic release of nascent polypeptides from ribosomes by cleaving the ester bond of peptidyl-tRNA . Key features include:
-
Codon Specificity: RF1 terminates translation at UAG and UAA, while RF2 (encoded by prfB) recognizes UAA and UGA .
-
Structural Domains: RF1’s N-terminal domain is critical for codon recognition, while the C-terminal domain mediates peptidyl-tRNA hydrolysis .
Deletion of prfA eliminates competition between RF1 and orthogonal tRNAs at UAG codons, enabling efficient incorporation of non-standard amino acids (NSAAs) .
Development of Recombinant prfA
Recombinant RF1 is produced in E. coli for research and industrial applications. Product specifications from Cusabio (CSB-EP015197ENV.20) include :
| Property | Detail |
|---|---|
| Molecular Weight | 47.4 kDa |
| Purity | >95% (SDS-PAGE) |
| Host | E. coli |
| Applications | Enzyme activity studies |
This recombinant protein retains native functionality, making it valuable for studying translation mechanics and engineering termination systems .
Applications in Cell-Free Protein Synthesis (CFPS)
Genomically recoded E. coli strains lacking RF1 (ΔprfA) enhance CFPS yields of NSAA-containing proteins. Key findings include:
Performance Comparison: RF1-Present vs. RF1-Deficient Systems
| Parameter | RF1+ (μg/mL) | RF1– (μg/mL) | Improvement |
|---|---|---|---|
| Active sfGFP132pPaF | 71 ± 6 | 190 ± 20 | 250% |
| Full-Length Protein Ratio | 20% | 78% | 3.9x |
| Data derived from S30 extracts of rEc.E13.ΔprfA . |
Removing RF1 reduces truncated protein products, as RF1 competes with NSAA-charged tRNAs at UAG codons . CFPS systems using RF1-deficient strains outperform reconstituted PURE systems in cost ($0.05 vs. $10.00 per reaction) and yield (190 μg/mL vs. 41 μg/mL) .
Genomic Engineering and Adaptive Evolution
Knocking out prfA enables UAG codon reassignment for synthetic biology:
-
Strain Optimization: rEc.E13.ΔprfA (with 13 genomic UAG→TAA recodings) achieves 53% NSAA suppression efficiency, compared to 21% in RF1-positive strains .
-
Fitness Costs: RF1 deletion slows growth (doubling time increases from 47 to 65 minutes in 2xYTPG media), but further genomic edits (e.g., csdA and endA deletions) improve CFPS yields to 1,300 μg/mL .
Adaptive evolution experiments reveal compensatory mutations in prfB (RF2) and prfC (RF3) in ΔprfA strains, stabilizing translation termination efficiency .
Industrial and Research Implications
Recombinant RF1 and RF1-deficient systems are pivotal for:
Product Specs
Target Background
KEGG: ecm:EcSMS35_1931
Q&A
What is Peptide Chain Release Factor 1 and what gene encodes it in E. coli?
Peptide Chain Release Factor 1 (RF1) is a critical protein involved in translation termination in Escherichia coli. It is encoded by the prfA gene and functions by recognizing the stop codons UAA and UAG (amber) to terminate protein synthesis . RF1 plays a fundamental role in the cellular translation machinery by facilitating the release of newly synthesized polypeptide chains from the ribosome when a stop codon is encountered.
What is the relationship between RF1 and RF2 in E. coli?
RF1 and RF2 have partially overlapping functions in E. coli, with distinct stop codon recognition patterns:
-
RF1 recognizes UAA and UAG stop codons
-
RF2 recognizes UAA and UGA stop codons
Which E. coli strains permit successful RF1 knockout?
The ability to knock out RF1 varies significantly among E. coli strains, depending primarily on their RF2 genotype:
| Strain Category | Specific Strains | RF2 Status | RF1 Knockout Feasibility |
|---|---|---|---|
| E. coli B strains | REL606, BL21, BL21(DE3) | Wild-type (Ala246) | Successful (yielding strains CW1.0, CW2.0, JX1.0) |
| E. coli K-12 strains | MG1655, DH10β, HT115 | Mutant (Thr246) | Unsuccessful |
| Modified K-12 | DH10βf (with reverted RF2) | Reverted to wild-type (Ala246) | Successful |
This strain-dependent pattern directly correlates with RF2 functionality, demonstrating that wild-type RF2 is sufficient for cell viability in the absence of RF1 .
What is the recommended methodology for creating RF1 knockout strains?
Based on successful experimental approaches, the following methodology is recommended for RF1 knockout:
-
Strain selection: Choose E. coli B strains with wild-type RF2 (containing Ala246) or modify K-12 strains to revert the A246T mutation in RF2.
-
Knockout procedure: Employ λ red recombinase-based homologous recombination to replace the prfA gene with a selection marker such as the chloramphenicol acetyltransferase gene .
-
Verification: Confirm successful knockout through genomic PCR amplification of the prfA locus, where the band size will differ between wild-type and knockout strains .
-
Phenotypic assessment: Evaluate growth characteristics, as RF1 knockout strains typically exhibit longer doubling times, especially when additional genetic modifications for unnatural amino acid incorporation are introduced .
How does the A246T mutation in RF2 affect RF1 knockout feasibility?
The A246T mutation in RF2, present in E. coli K-12 strains, reduces its release activity for UAA stop codons by approximately 5-fold . Since UAA is responsible for terminating translation in about 64% of E. coli genes, this mutation creates a critical dependency on RF1 for proper translation termination.
The mechanistic explanation is that when RF1 is absent in strains with the A246T mutation in RF2, the mutated RF2 cannot efficiently recognize all UAA stop codons, leading to widespread translation termination defects that are incompatible with cell viability. Reverting this mutation to the wild-type Ala246 in K-12 strains restores efficient UAA recognition by RF2, making RF1 dispensable .
How does RF1 knockout affect cell growth and viability?
RF1 knockout has measurable effects on cellular growth dynamics:
| Strain | Condition | Doubling Time (min) | Notes |
|---|---|---|---|
| BL21(DE3) (wild-type) | Standard | 41 | Baseline growth |
| JX1.0 (RF1 knockout) | Standard | 135 | ≈3× longer doubling time |
| JX1.0 | With tRNA^CUA^Tyr/LW1RS + pActF | 253 | ≈6× longer than wild-type |
| BL21(DE3) | With tRNA^CUA^Tyr/LW1RS + pActF | 41 | No significant change |
These data demonstrate that RF1 knockout alone causes moderate growth defects, which are further exacerbated when machinery for unnatural amino acid incorporation is introduced . This growth defect likely reflects the metabolic burden of proteome-wide UAG reassignment and possible translation inefficiencies.
What happens to UAG codons in RF1 knockout strains?
In RF1 knockout strains, UAG codons no longer function efficiently as translation termination signals since the cellular machinery responsible for recognizing them has been removed. This creates two significant consequences:
-
Near-random amino acid incorporation: Without supplementation, endogenous near-cognate tRNAs may incorporate various amino acids at UAG positions with low efficiency.
-
Targeted reassignment: When supplemented with an orthogonal tRNA/aminoacyl-tRNA synthetase pair designed to recognize UAG, these codons can be systematically reassigned to encode specific natural or unnatural amino acids .
The reassignment is "unambiguous" in RF1 knockout strains because there is no competition between termination and amino acid incorporation at UAG codons, leading to more efficient incorporation of the desired amino acid .
How can RF1 knockout strains enhance unnatural amino acid incorporation efficiency?
RF1 knockout strains provide significant advantages for unnatural amino acid (Uaa) incorporation through several mechanisms:
-
Elimination of termination competition: Removing RF1 eliminates the competition between termination and suppression at UAG codons, allowing more efficient incorporation of Uaas .
-
Multi-site incorporation: RF1 knockout permits efficient incorporation of Uaas at multiple UAG sites within the same protein, which is typically difficult in wild-type strains due to termination competition .
-
Lower suppressor tRNA requirements: The absence of RF1 competition means that lower levels of suppressor tRNA expression can achieve efficient incorporation, reducing cellular stress .
A direct comparison demonstrates that when expressing an orthogonal tRNA^CUA^Tyr/LW1RS pair with p-acetylphenylalanine (pActF), the RF1 knockout strain (JX1.0) shows dramatically slower growth compared to its parent strain (BL21(DE3)), suggesting more efficient incorporation of pActF at multiple UAG positions throughout the proteome .
What orthogonal translation systems work effectively in RF1 knockout strains?
Several orthogonal tRNA/synthetase pairs have been demonstrated to work effectively in RF1 knockout strains for UAG reassignment:
The key advantage of RF1 knockout strains is their ability to incorporate unnatural amino acids at multiple sites within the same protein without competition from release factors, making them valuable for complex protein engineering applications .
What are the evolutionary implications of RF1 dispensability?
The dispensability of RF1 in E. coli has significant evolutionary implications:
-
Genetic code plasticity: The viability of RF1 knockout strains demonstrates that the genetic code is more adaptable than previously thought, supporting theories about code evolution and reassignment .
-
Natural precedents: Stop codon reassignment has occurred naturally in certain organisms, and RF1-knockout E. coli provides a model system to study similar evolutionary processes .
-
Genome recoding potential: The ability to remove RF1 and reassign UAG codons suggests possibilities for large-scale genome recoding, potentially creating organisms with novel genetic codes resistant to viral infection or with enhanced biosynthetic capabilities .
RF1 knockout strains provide a unique experimental platform for studying fundamental questions about the evolution of the genetic code and the minimal requirements for translation termination in living organisms.
How can researchers address potential proteome-wide effects of UAG reassignment?
When working with RF1 knockout strains, researchers should consider several strategies to address proteome-wide effects of UAG reassignment:
-
Genomic analysis: Before knockout, analyze the strain's genome to identify all genes terminating with UAG and assess potential functional consequences of extended proteins.
-
Complementation strategies: For essential genes terminating with UAG, consider site-directed mutagenesis to change UAG to alternative stop codons (UAA or UGA) before RF1 knockout.
-
Growth optimization: Develop optimized media compositions and growth conditions to mitigate the growth defects associated with RF1 knockout and UAG reassignment .
-
Inducible systems: Consider implementing inducible orthogonal translation systems to control the timing and extent of UAG reassignment, minimizing cellular stress.
-
Proteomic analysis: Employ comparative proteomics to characterize changes in protein expression, modification, and truncation patterns resulting from RF1 knockout.
These approaches can help manage the complex consequences of proteome-wide UAG reassignment while maximizing the utility of RF1 knockout strains for specific research applications.
Why might RF1 knockout attempts fail in certain E. coli strains?
Failed RF1 knockout attempts may result from several factors:
-
RF2 mutation status: The primary reason for failure is the presence of the A246T mutation in RF2, which reduces its UAA recognition efficiency by approximately 5-fold . Before attempting RF1 knockout, sequence the RF2 gene to determine if this mutation is present.
-
Strain background: E. coli K-12 strains typically carry the A246T mutation in RF2 and are not amenable to direct RF1 knockout without prior modification . B strains with wild-type RF2 are preferred.
-
Recombination efficiency: Low λ red recombinase expression or activity can reduce knockout efficiency. Ensure proper induction of the recombination system before attempting knockout.
-
Essential gene considerations: In some experimental conditions or genetic backgrounds, genes terminating with UAG may become functionally essential, preventing viable RF1 knockout.
If knockout attempts fail, consider reverting the A246T mutation in RF2 to Ala246 before attempting RF1 knockout, as demonstrated successfully with the DH10βf strain .
How can the growth defects in RF1 knockout strains be minimized?
Growth defects in RF1 knockout strains can be minimized through several approaches:
-
Media optimization: Supplement growth media with components that reduce cellular stress, such as additional amino acids or osmoprotectants.
-
Temperature adjustment: Lower growth temperatures (30°C instead of 37°C) may reduce proteotoxic stress from misfolded proteins resulting from UAG readthrough.
-
Controlled expression: Use tightly regulated promoters for expressing orthogonal tRNA/synthetase pairs to prevent excessive suppression burden.
-
Adaptive evolution: Continuous culture of RF1 knockout strains can select for compensatory mutations that improve growth while maintaining the desired genotype.
-
Selective UAG reassignment: When possible, engineer genes of interest to contain UAG codons while converting endogenous UAG stop codons to alternative stop codons, reducing proteome-wide effects.
These strategies can help balance the research benefits of RF1 knockout strains with the need for reasonable growth characteristics in experimental settings.
