Genetic code expansion inserts a non-canonical amino acid (ncAA) at a chosen site in a protein by recoding one codon — usually the amber stop codon UAG — as a sense codon. Two molecular species race for every ribosome that reaches that UAG:
Orthogonal pair: aaRS + ncAA + tRNA_CUA → ncAA-tRNA_CUA (charging, Michaelis-Menten)
occupancy = [ncAA] / (Km + [ncAA]), Km ≈ 0.4 mM
Kinetic partitioning at the UAG codon (competing exponential arrivals):
k_supp = k_supp,max · (tRNA level) · occupancy (successful readthrough)
k_term = k_term,max · (RF1 level) (premature termination)
P(readthrough) = k_supp / (k_supp + k_term)
Each ribosome event draws its own random race:
t_supp ~ Exponential(k_supp), t_term ~ Exponential(k_term)
winner = min(t_supp, t_term) → same long-run frequency as P(readthrough) above
When the charged orthogonal tRNA wins the race, the ribosome reads through UAG, inserts the ncAA, and continues to the natural stop codon — yielding a full-length protein carrying a genetically encoded chemical handle (e.g. an azide or alkyne for click-chemistry bioconjugation). When RF1 wins, translation terminates early and a truncated, usually non-functional peptide is released.
- ncAA concentration — raises the fraction of orthogonal tRNA charged (Michaelis-Menten occupancy).
- Orthogonal tRNA/aaRS expression — scales how much charged suppressor tRNA is available to compete.
- RF1 activity — wild-type E. coli has strong RF1; genome-recoded, RF1-attenuated or ∆RF1 chassis strains (e.g. C321.∆A) remove UAG entirely from the genome and knock out RF1, dramatically raising suppression efficiency.
- Translation initiation rate — sets ribosome throughput on the transcript, which scales how fast UAG-race events accumulate.
This 2D view runs the exact same stochastic per-ribosome race as the 3D version, drawn as ribosomes sliding along an mRNA track. Watch the measured full-length yield (an empirical frequency over many independent races) converge on the theoretical k_tRNA/(k_tRNA+k_RF1) ratio — and confirm that dropping RF1 activity (e.g. the RF1-attenuated chassis button) genuinely raises it.