Recombinant Peptide chain release factor 1 (prfA)

What is the basic structure of RF1 and how does it differ from RF2?

RF1 is composed of four domains with approximate dimensions of 75 Å × 70 Å × 45 Å. The crystal structure of RF1 from Thermotoga maritima, determined at 2.65 Å resolution using selenomethionine single-wavelength anomalous dispersion (SAD) techniques, reveals that while RF1 shares similar domain topology with E. coli RF2, there are notable differences . The N-terminal domain of RF1 is shorter than that of RF2, while its C-terminal domain is longer .

Additionally, RF1 exhibits a rigid-body movement in its N-terminal domain relative to RF2, with an angle of approximately 90 degrees . A key distinguishing feature is that RF1 contains a tripeptide anticodon PVT motif instead of the SPF motif found in RF2, which confers specificity towards different stop codons . The dynamic movement of domains I and III, anchored to the central domain by hinge loops, appears to be an intrinsic property necessary for proper function with the ribosome .

How does the 5' flanking codon influence RF1-mediated translation termination?

The codon preceding a stop codon (5' flanking codon) significantly influences translation termination efficiency. Research shows that isocodons (codons that differ only in the last base but are decoded by the same tRNA species) affect termination at UAG differently in strains with mutant or wild-type RF1 .

The nature of the wobble base anticodon-codon interaction at the ribosomal peptidyl-tRNA binding site (P-site) appears to influence RF1 sensitivity . For some isoaccepting P-site tRNAs (such as tRNA3Pro versus tRNA2Pro and tRNA4Thr versus tRNA1,3Thr), the effect differs between mutant and wild-type RF1, suggesting an interaction between RF1 at the aminoacyl-tRNA acceptor site (A-site) and the P-site tRNA itself .

The glycine codons GGA and GGG at the ribosomal P-site are associated with almost threefold higher readthrough of UAG than any of the other 42 codons tested in strains with wild-type RF1, but this differential response is lost in strains with mutant RF1 . This indicates specific codon-anticodon interactions play critical roles in termination efficiency.

How can RF1 deletion enhance non-standard amino acid incorporation in recombinant protein synthesis?

RF1 deletion (ΔprfA) substantially enhances the incorporation of non-standard amino acids (NSAAs) at amber stop codons. In cell-free protein synthesis (CFPS) systems developed from genomically recoded E. coli strains lacking RF1, the efficiency of incorporating non-standard amino acids such as p-propargyloxyphenylalanine (pPaF) increased dramatically compared to systems with RF1 present .

Specifically, extracts from RF1-deficient strains (rEc.E13.ΔprfA) synthesized active sfGFP132pPaF at yields of 190 ± 20 μg/mL compared to only 71 ± 6 μg/mL in RF1-present extracts, representing a 2.5-fold increase in yield and suppression efficiency (53% versus 21%) . The table below illustrates these comparative yields:

The key advantage of RF1 deletion is that the orthogonal tRNA (o-tRNA) need only compete against natural suppression mechanisms rather than against RF1, resulting in a higher proportion of full-length protein (78% in RF1-deficient versus 20% in RF1-present systems) .

What methodological approach should be used to develop a viable RF1-deficient strain for protein synthesis applications?

Developing a viable RF1-deficient strain requires careful genomic recoding to address essential genes containing amber (TAG) stop codons. The research data suggests the following methodological approach:

• First identify essential genes containing amber stop codons that would be affected by RF1 deletion. In the case of the rEc.E13.ΔprfA strain, researchers recoded 13 essential genes containing amber codons (including coaD, hemA, mreC, murF, lolA, lpxK, yafF, pgpA, sucB, fabH, fliN, and atpE) .

• Replace the amber (TAG) codons with alternative stop codons (typically TAA) to ensure proper termination in the absence of RF1.

• Introduce an antibiotic resistance marker (such as spectinomycin resistance gene) into the prfA locus to create the RF1 knockout strain and allow for selection .

• Evaluate growth phenotypes, as RF1 deletion typically affects growth rates. The rEc.E13.ΔprfA strain had a doubling time of 64.6 ± 0.9 min compared to 47 ± 2 min for the parent rEc.E13 strain in 2xYTPG media .

• Optimize culture conditions to improve the growth of RF1-deficient strains, which generally exhibit slower growth compared to parent strains due to incomplete recoding of the genome .

What control experiments should be included when investigating RF1-mediated translation termination efficiency?

When investigating RF1-mediated translation termination, several critical controls should be included:

How should researchers quantify and analyze readthrough efficiency in RF1 studies?

To properly quantify and analyze readthrough efficiency in RF1 studies, researchers should employ multiple complementary approaches:

• Fluorescence-based assays: For fluorescent reporter proteins like sfGFP, measure active protein yield through fluorescence intensity and calculate suppression efficiency as the ratio of fluorescence in the experimental condition to that of the wild-type control without a stop codon .

• Radiolabeling assays: Incorporate radioactive amino acids (such as 14C-leucine) into newly synthesized proteins and visualize full-length and truncated products using autoradiography . This allows calculation of the ratio between full-length and truncated products.

• Total protein quantification: Use trichloroacetic acid precipitation of radiolabeled proteins to quantify total protein synthesis and compare results between different experimental conditions .

• Statistical analysis: Apply appropriate statistical tests to determine significance of differences between conditions. Data should be reported with standard deviations from multiple independent experiments (typically n≥3) .

• Dose-response analysis: When examining factors that affect readthrough (such as RF1 concentration), use varying concentrations to establish dose-response relationships . For example, the research shows that increasing RF1 amounts in a PURE reaction system leads to decreased full-length modified protein and increased truncated protein .

How can researchers reconcile contradictory findings about codon context effects on RF1 function?

When facing contradictory findings regarding codon context effects on RF1 function, researchers should:

• Examine strain backgrounds: Different E. coli strains may have varying levels of suppressor tRNAs or modifications in translation machinery that affect termination efficiency. Compare the genetic backgrounds used in conflicting studies .

• Consider growth conditions: Translation termination efficiency can be affected by growth phase and media composition, which influence tRNA abundance and modification status. Standardize growth conditions for meaningful comparisons .

• Analyze experimental systems: Cell-based versus cell-free systems may yield different results. In cell-free systems like PURE, components can be individually controlled, whereas cellular studies involve additional regulatory factors .

• Examine the presence of competing factors: The presence or absence of competing factors (such as suppressor tRNAs or RF1 itself) dramatically changes the outcome of termination events at stop codons .

• Consider sequence context beyond the immediate P-site codon: Examine whether studies considered the same sequence context range. Some effects may depend on interactions spanning more than just the codon immediately preceding the stop codon .

• Analyze protein product completely: Use methods that can detect both full-length and truncated products (like autoradiography) rather than only measuring active protein, which could miss important effects on termination .

What are the key considerations when comparing RF1 function across different experimental systems?

When comparing RF1 function across different experimental systems, consider:

• System composition: Cell-free extract-based systems contain many cellular components that might influence RF1 function, while purified systems (like PURE) contain only defined components. The research shows that S30 extract-based approaches produce about 5 times more protein (190 ± 20 μg/mL) than the PURE translation system (41 ± 3 μg/mL) when lacking RF1 .

• RF1 concentration: In natural cells and crude extracts, RF1 concentration is not precisely controlled, while in reconstituted systems, researchers can add defined amounts of RF1. Studies show that as RF1 concentration increases, full-length modified protein production decreases while truncated protein increases .

• tRNA competition: The ratio of RF1 to competing tRNAs (including suppressor tRNAs) varies between systems and affects apparent termination efficiency .

• Temperature effects: RF1 from thermophilic organisms (like Thermotoga maritima) may have different structural properties and temperature optima compared to mesophilic counterparts .

• Structural dynamics: RF1 exhibits dynamic movements between domains that are important for function . Different experimental conditions may affect these dynamics.

• Cost-effectiveness analysis: Consider not only performance but also economic factors when choosing systems. Extract-based CFPS is significantly more cost-effective (less than $0.05/reaction) compared to PURE systems (more than $10.00/reaction) .

What are promising research applications of RF1-deficient systems in synthetic biology?

RF1-deficient systems offer several promising research applications in synthetic biology:

• Expanded genetic code: RF1-deficient strains allow more efficient incorporation of non-standard amino acids at amber codons, enabling the creation of proteins with novel chemical functionalities for various applications including biocatalysis, biotherapeutics, and materials science .

• Multi-site NSAA incorporation: The improved suppression efficiency in RF1-deficient systems (53% vs. 21% in RF1-present systems) makes incorporating NSAAs at multiple sites more feasible, expanding the repertoire of possible protein modifications .

• Cell-free synthetic biology platforms: RF1-deficient extracts provide high-yield platforms for synthesizing proteins containing NSAAs without cellular viability constraints. These platforms could be used for rapid prototyping of novel proteins and pathways .

• Evolutionary studies: RF1-deficient strains could serve as starting points for directed evolution experiments to expand the genetic code or evolve novel translation properties.

• Studying translation termination mechanisms: RF1-deficient systems provide a clean background for introducing modified or engineered release factors to study the fundamental mechanisms of translation termination .

How might structural data on RF1 inform the design of engineered release factors with novel properties?

Structural data on RF1 can guide the design of engineered release factors with novel properties through several approaches:

• Anticodon motif engineering: The tripeptide PVT motif in RF1 determines stop codon specificity . Engineering this region could create modified release factors with altered or expanded codon recognition properties.

• Domain movement optimization: RF1 exhibits dynamic movements between domains I and III, which are anchored to the central domain by hinge loops . Engineering these hinge regions could modify the conformational dynamics of RF1 to enhance or alter its function.

• Interface engineering: Understanding the structural interfaces between RF1 and the ribosome could enable the design of release factors with altered ribosome binding properties, potentially creating orthogonal translation systems.

• P-site tRNA sensitivity engineering: RF1 shows sensitivity to the nature of the codon-anticodon interaction at the P-site . Engineering the regions that interact with the P-site tRNA could create release factors with modified context sensitivities.

• Nascent chain interaction engineering: The C-terminal amino acid of the nascent peptide affects termination efficiency, with high α-helix propensity correlating with increased termination . Engineering RF1 to modify these interactions could create release factors with novel properties.

The crystal structure of RF1 from Thermotoga maritima at 2.65 Å resolution provides atomic-level details to guide these engineering efforts , while functional studies in various genetic backgrounds provide insights into which modifications might be most effective .