RESUMO
Organisms possessing genetic codes with unassigned codons raise the question of how cellular machinery resolves such codons and how this could impact horizontal gene transfer. Here, we use a genomically recoded Escherichia coli to examine how organisms address translation at unassigned UAG codons, which obstruct propagation of UAG-containing viruses and plasmids. Using mass spectrometry, we show that recoded organisms resolve translation at unassigned UAG codons via near-cognate suppression, dramatic frameshifting from at least -3 to +19 nucleotides, and rescue by ssrA-encoded tmRNA, ArfA, and ArfB. We then demonstrate that deleting tmRNA restores expression of UAG-ending proteins and propagation of UAG-containing viruses and plasmids in the recoded strain, indicating that tmRNA rescue and nascent peptide degradation is the cause of impaired virus and plasmid propagation. The ubiquity of tmRNA homologs suggests that genomic recoding is a promising path for impairing horizontal gene transfer and conferring genetic isolation in diverse organisms.
Assuntos
Códon de Terminação/genética , Proteínas de Escherichia coli/genética , Transferência Genética Horizontal/genética , Código Genético/genética , Proteínas de Ligação a RNA/genética , Escherichia coli/genética , Mudança da Fase de Leitura do Gene Ribossômico/genética , Regulação Bacteriana da Expressão Gênica , Genoma Bacteriano/genética , Plasmídeos/genética , RNA Bacteriano/genética , Vírus/genéticaRESUMO
Horizontally transferred genetic elements such as viruses and conjugative plasmids move DNA between organisms, increasing genetic diversity but destabilizing engineered biological systems. Here, we used a genomically recoded Escherichia coli strain lacking UAG stop codons and the recognition protein release factor 1 to study how an alternative genetic code influences horizontally transferred genetic element propagation. The alternative genetic code conferred resistance to multiple viruses (λ, M13, P1, MS2) at titers up to 10(11) PFU/ml and impaired conjugative plasmids (F and RK2) up to 10(5)-fold. By recoding UAG codons to UAA in viruses and plasmids, we restored viral infectivity and conjugative function. Propagating viruses on a mixed community of cells with standard and alternative genetic codes reduced viral titer, and over time viruses adapted to the alternative genetic code. This work demonstrates that altering the genetic code broadly obstructs the propagation of horizontally transferred genetic elements and supports the use of genomic recoding as a strategy to stabilize engineered biological systems.