RF1 is Nonessential

Release factor one (RF1) is nonessential in E. coli

Natural code evolution occurs over millions of years. Extant organisms harboring altered genetic codes are at the end-point of the code evolution, and records for the process had been buried in the recess of evolution. To enable in-depth investigation of code change and any concurrent cellular adaptations in real time, it is necessary to generate a model organism that is able to undergo such evolutionary processes in the laboratory. Eukaryotes and archaea use a single release factor (RF) to recognize all three stop codons, but bacteria use two: RF1 for UAA/UAG and RF2 for UAA/UGA. Why are there two RFs in bacteria whereas a single RF is sufficient for the other two domains of life?

Synthetically recoding a genome may afford new properties to the organism through encoding Uaas, which demands target codons to be reassigned in high efficiency and without ambiguity. An attractive route is to reassign the amber stop codon UAG in bacteria to instead encode a Uaa. Orthogonal tRNA-synthetase pairs have been engineered to incorporate Uaas in response to UAG, yet the presence of RF1 makes the meaning of UAG ambiguous, being both a stop signal and a Uaa simultaneously. RF1 competition limits the incorporation of Uaas at a single UAG site with low efficiency; such low UAG-encoding efficiency prevents effective use of Uaas at multiple sites to explore novel protein and organismal properties through directed evolution.

We aimed to fully reassign the UAG codon to a sense codon, for which an imperative step is to knock out RF1 from the E. coli genome. However, no free-living bacterium has been found lacking either RF1 or RF2. For E. coli, RF1 has been reported to be essential since the 1980s. Contradictory to this paradigm, we discovered that 
RF1 could be unconditionally knocked out from various
 E. coli stains. The apparent essentiality of RF1 was found to be caused by 
the inefficiency of a mutant RF2 in terminating all UAA stop codons.


Figure 3.1: The RF1-knockout strain JX33 enables unnatural amino acids to be incorporated at multiple sites (indicated by surrounding numbers) with high efficiency. Left half, common E. coli; right half, JX33.

We have thus generated the first series of E. coli strains with RF1 knocked out unconditionally. We then demonstrated that these strains allowed Uaa incorporation at the UAG codon with high efficiency, and more importantly, enabled simultaneous incorporation of Uaa at multiple sites. These unprecedented properties make the RF1 knockout bacterium a unique host to efficiently harness Uaa in the same manner as natural amino acids for evolving new protein functions and biological properties. Knockout of RF1 provides novel insights on the evolution of RFs, affords a previously unavailable model for studying code evolution, and enables new research of genome recoding and code engineering for synthetic biology.