JX33 is a novel RF1 knockout strain with unique properties for Uaa incorporation. Generated 3.5 years ago, JX33 has been stable and autonomous without major growth or deleterious defects. Without RF1 competition, JX33 increases the amino acid incorporation efficiency at a single TAG site more than 100% than the parental RF1-containing JX2.0. More important, JX33 enables amino acids to be incorporated at multiple TAG sites without decreasing efficiency. This ability has not been achieved in any cells before, and is an essential trait for UAG being completely reassigned as a sense codon.
Although RF1 competition no longer exists in JX33, the efficiency of UAG functioning as a sense codon depends on multiple factors. Orthogonal synthetases evolved for Uaas are often less active than wild type synthetases5,6
, and thus may generate less aminoacylated orthogonal tRNAs. In addition, the binding of natural aminoacyl-tRNAs to elongation factor Tu and to ribosome has been evolutionary tuned for optimal decoding33,34
, yet the Uaa loaded orthogonal tRNA has not been fully optimized toward either. Moreover, many tRNAs are subjected to post-transcriptional modifications for specific and efficient decoding of cognate codons35
. The orthogonal tRNA, with its anticodon artificially mutated to CUA, has not evolutionary optimized for UAG decoding. All these factors could make the UAG codon less efficient than canonical sense codons in encoding amino acids. For these reasons, the four UAG codons placed closely in the N-terminus of the H3a may behave as a cluster of “rare” codons. In E. coli
, a rare-codon cluster lowers protein expression level36
, which may account for the drop-off in yields with additional UAG observed for H3a expression. These non-optimal factors would also explain why Uaa-containing mutant proteins have not yet reached the same expression level as the wt protein.
A long-standing question for genetically encoding Uaas with a stop codon is how endogenous genes ending with the stop codon are affected. By studying two representative endogenous genes, we found that an amber suppressor tRNA/synthetase did not efficiently incorporate its cognate amino acid at the legitimate TAG site in the presence of RF1. This surprising finding suggests that an unknown mechanism may prevent these legitimate stop codons from being suppressed. The inefficient suppression of endogenous TAGs in JX2.0 explains why no significant adverse effect to E. coli is observed when orthogonal amber suppressor tRNA/synthetase pairs are used to incorporate Uaas. However, we discovered that upon RF1 removal in JX33 the TAG codon of endogenous genes was efficiently suppressed by the tRNA/synthetase pair. Translation then extended to the next in-frame different stop codon when there is no transcription terminator before the next stop codon; when there is a transcription terminator, translation was terminated between the TAG and the mRNA end defined by the terminator hairpin. Endogenous UAG suppression in JX33 also led to a slower growth phenotype. Studying UAG suppression in more endogenous genes would confirm whether the above observations are general.
RF1 is reported to be essential for E. coli, but our results argue against this paradigm. We showed that RF1 can be knocked out when wild type RF2 expression is not auto-regulated. The resultant JX31 strain has no compensatory mutations anywhere in the genome. Although JX31 has a slower growth rate, it is an independent and stable strain with RF1 deleted, which suggests that RF1 is nonessential for E. coli. Interestingly, the A293E mutation of RF2 found in JX33 restores the growth rate of JX33 to the same level of the parental JX2.0. However, the RF2(A293E) is unable to substitute RF1 in terminating UAG codons because it could not rescue the RF1 temperature sensitive phenotype in MRA8 cells. We note that non-stop incorporation of pActF into EGFP was also observed in JX31, which has no RF2(A293E) mutation; the protein yields for 1-, 2- and 3-TAG EGFP mutants from JX31 were 5.7 (±0.4), 6.1 (±0.5) and 7.0 (±0.5) mg/L, respectively. This result suggests that the RF2(A293E) mutation is not required for efficient incorporation of Uaa at multiple TAG sites. Nonetheless, how A293E mutation contributes to the fast growth of JX33 warrants further studies.
When this paper was being prepared, it was reported that RF1 can be knocked out after supplying 7 essential genes and a suppressor tRNA on a plasmid37
. The dependence on simultaneous change of many essential genes is consistent with the previous conclusion that RF1 is essential. A major difference of our work is that the knockout of RF1 is independent of supplying any other genes or a suppressor tRNA. More important, the generation of JX31 suggests that RF1 is nonessential. To our best knowledge, this work represents the first unconditional knockout of RF1 and the generation of an autonomous stable RF1 deletion strain.
Selective incorporation of Uaas at multiple sites will open up new possibilities in protein research and laboratory evolution. For instance, multisite incorporation of posttranslational modification mimics (e.g., ActK and pCmF) will be valuable for studying epigenetics and signal transductions. JX33 may enable laboratory evolution of bacteria in search for new protein properties or organismal functions by exploiting the novel properties of Uaas. Such experiments were not feasible or effective before, as the Uaa is incorporated at a single site with low efficiency; novel functions may require an Uaa at multiple positions simultaneously. Moreover, the presence of RF1 generates truncated protein products, which may interfere with selection and evolution. JX33 resolves all these problems and should prove valuable in harnessing the expanding Uaa repertoire for directed evolution.
RF1 knockout strains can also be valuable for investigating the evolution of the genetic code. Organisms in different taxa have been found to reassign stop codons to sense codons1,2
, yet they represent the reassignment endpoint and provide limited information on the reassignment process and organismal adaptation. This study demonstrates the feasibility of reassigning the UAG stop codon to a sense codon in the extant organism E. coli
, providing empirical evidence in support of such codon reassignment events. E. coli
is tolerant of codon reassignment and unexpectedly flexible in adapting to a new code, suggesting that the code can evolve in modern organisms. JX3.0 affords a previously unavailable model system for experimentally studying the physiological change and adaptation of a living organism to codon reassignment on a laboratory time scale.