Usually, DNA passes from parent to offspring, vertically down the generations. But not always. In some cases, it can move directly from one organism to another by a process called horizontal gene transfer. In bacteria, this happens when DNA segments pass through a bacterium’s cell wall, which can then be picked up by another bacterium. Because the vast majority of organisms share the same genetic code, the bacteria can read this DNA with ease, as it is in the same biological language.

Horizontal gene transfer helps bacteria adapt and evolve to their surroundings, letting them swap and share genetic information that could be useful. The process also poses a threat to human health because the DNA that bacteria share can help spread antibiotic resistance. However, some organisms use an alternative genetic code, which obstructs horizontal gene transfer. They cannot read the DNA transmitted to them, because it is in a different ‘biological language’. The mechanism of how this language barrier works has been poorly understood until now.

Ma, Hemez, Barber et al. investigated this using Escherichia coli bacteria with an artificially alternated genetic code. In this E. coli, one of the three-letter DNA ‘words’ in the sequence is a blank – it does not exist in the bacterium’s biological language. This three-letter DNA word normally corresponds to a particular protein building block. Using a technique called mass spectrometry, Ma et al. analyzed the proteins this E. coli forms. The results showed that it has several strategies to deal with DNA transmitted horizontally into the bacterium. One method is destroying the proteins that are half-created from the DNA, using molecules called tmRNAs. These are part of a rescue system that intervenes when protein translation stalls on the blank word. The tmRNAs help to add a tag to half-formed proteins, marking them for destruction.

This mechanism creates a ‘genetic firewall’ that prevents horizontal gene transfer. In organisms engineered to work from an altered genetic code, this helps to isolate them from outside interference. The findings could have applications in creating engineered bacteria that are safer for use in fields such as medicine and biofuel production.

The standard genetic code allows faithful translation of proteins across nearly all living organisms and enables horizontally transferred genetic elements (HTGEs), such as conjugative plasmids and viruses, to exploit a host’s translational machinery (Krakauer and Jansen, 2002). Since naturally occurring exceptions to the standard genetic code exist (Ambrogelly et al., 2007; Knight et al., 2001), researchers have hypothesized that such alternative genetic codes might arise to escape viral predation (Shackelton and Holmes, 2008). Recent research supports this hypothesis, with modification to codon usage or the genetic code reducing the ability of viruses and conjugative plasmids to exploit their hosts (Coleman et al., 2008; Lajoie et al., 2013b; Ma and Isaacs, 2016). Given the medical, technological, and evolutionary importance of HTGE-mediated horizontal gene transfer (HGT) (Davies, 1994; Gogarten and Townsend, 2005; Moe-Behrens et al., 2013; Ochman et al., 2000), understanding the molecular basis for how alternative genetic codes impede HTGEs is vital.

At the molecular level, an alternative genetic code arises from reassignment of one or more codons in the genetic code, which stems from a change in the ability of an aminoacyl-tRNA or release factor (RF) to recognize codon(s) during translation. One possible alteration of the genetic code is the loss of a codon assignment through the deletion or modification of an aminoacyl-tRNA or release factor, removing the cell’s ability to decode that codon (Figure 1A). Such unassigned codons are found in alternative genetic codes in nature (Knight et al., 2001) and have been engineered into genomically recoded organisms (GROs) derived from Escherichia coli (Isaacs et al., 2011; Lajoie et al., 2013b). We recently demonstrated that a GRO with an unassigned UAG codon (i.e. lacking all instances of the UAG codon and release factor 1, RF1) impaired the propagation of HTGEs carrying UAG-ending genes, illustrating that alternative genetic codes can obstruct HGT (Ma and Isaacs, 2016) and establishing the GRO as an ideal model to study the molecular mechanisms that act at unassigned codons to impair HTGEs.

Figure 1

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Encountering an unassigned codon during translation leads to ribosomal stalling, and without resolution, to cell death (Keiler and Feaga, 2014). However, the survival of organisms engineered to lack RF1 but retaining some UAG codons in their protein-coding sequences (Heinemann et al., 2012; Mukai et al., 2010) and the ability of GROs to resist exploitation by and continue growth in the presence of HTGEs (Ma and Isaacs, 2016) indicates that E. coli can resolve translation at unassigned UAG codons. We hypothesize that three mechanisms could resolve translation at prokaryotic ribosomes encountering these unassigned codons, each resulting in peptides with different C-terminal sequences (Figure 1B): (1) suppression of the codon by a near-cognate or mutated tRNA (e.g. amber suppressor) and continued translation, (2) frameshifting of bases along the mRNA transcript into a new reading frame and continued translation, or (3) stalling that elicits one of three ribosomal rescue pathways (tmRNA-SmpB, ArfA, or ArfB) in the cell (Keiler, 2015). The tmRNA-SmpB system acts as the primary rescue mechanism in prokaryotes, resolving ribosomal stalling that arises from the translation of mRNAs lacking a stop codon due to mRNA degradation, frameshifting, and stop codon read-through (Keiler, 2015). tmRNA-SmpB can also rescue ribosomes stalled on intact mRNAs for structural reasons (Cruz-Vera et al., 2011; Keiler, 2015; Li et al., 2012). The ssrA-encoded tmRNA associates with SmpB to form the tmRNA-SmpB complex, which adds a C-terminal degradation tag to peptides on stalled ribosomes (Tu et al., 1995). ArfA and ArfB, the secondary ribosomal rescue systems, alleviate stalling and release the stalled ribosome’s nascent peptide without modification (Chadani et al., 2012; Shimizu, 2012). tmRNA, ArfA, and ArfB all act on nonstop ribosomal complexes, which are stalled ribosomes that have reached the 3’ end of an mRNA because of stop-codon readthrough or because of the loss of a stop codon due to 3’ exonuclease degradation (Keiler, 2015). A possible fourth outcome identified from in vitro studies is loss of translational fidelity after the ribosome encounters rare or unassigned codons (Gingold and Pilpel, 2011), followed by untemplated termination by release factor 2 (RF2) (Zaher and Green, 2009).

Studies of ribosomal stalling arising at rare codons (Hayes et al., 2002) or in contexts of depleted or inefficient cognate decoding elements (George et al., 2016; Li et al., 2007; Roche and Sauer, 1999) suggest that a number of these mechanisms could resolve translation at unassigned codons, but a lack of well-characterized model organisms with an unassigned codon has precluded direct study of this question. Here, we use the GRO as a model to demonstrate that unassigned UAG codons in mRNA transcripts (1) elicit suppression, ribosomal frameshifting, and ribosomal rescue, (2) can induce ribosomal frameshifting from at least −3 to +19 nucleotides, and (3) lead to total loss of translational fidelity. By selectively deleting ribosomal rescue pathways in the GRO, we show that the tmRNA system is primarily responsible for rescuing ribosomes stalled at unassigned codons, with deletion of the tmRNA restoring expression of UAG-ending genes and re-enabling propagation of UAG-containing plasmids and viruses in the GRO. Our work reveals mechanistic details into how cells rescue ribosomes stalled at unassigned stop codons, providing insight into how alternative genetic codes act as barriers to HTGEs and demonstrating genomic recoding as a broadly applicable strategy to obstruct HGT in engineered organisms.

In this study, we use a genomically recoded organism (GRO) containing an unassigned UAG codon as a model to investigate the molecular mechanisms that obstruct the propagation of HTGEs in organisms with alternative genetic codes. We demonstrate that unassigned stop codons elicit near-cognate suppression, frameshifting, and the action of ribosomal rescue mechanisms (Figure 2). tmRNA-mediated ribosomal rescue prompted by the unassigned codon results in the degradation of nascent peptides translated from UAG-ending transcripts and obstructs the propagation of HTGEs (Figure 3, Figure 4). Additionally, ssrA deletion strains exhibit both significantly increased UAG-GFP yields (Figure 3C) and recovered propagation of HTGEs (Figure 4), consistent with evidence that deletion of ssrA removes inhibition of ArfA production and releases nascent peptides from stalled ribosomes without degradation (Chadani et al., 2011; Garza-Sánchez et al., 2011; Schaub et al., 2012). Our GRO model thus sheds light on the functional significance of previously described regulatory relationships while elucidating the unique mechanistic contributions of different ribosomal rescue systems in resolving translation at unassigned stop codons. These mechanistic outcomes that occur as a consequence of ribosomal stalling could be further investigated via ribosomal profiling in future work.

The mass spectrometry data collected from our GRO model demonstrate the striking proclivity for the ribosome to undergo un-programmed frameshifting at unassigned stop codons and represents, to our knowledge, the first in vivo study to examine such frameshifting. Prior studies have revealed programmed ribosomal frameshifting from −4 to +50 nucleotides (Atkins et al., 2016; Baranov et al., 2015; Huang et al., 1988; Yan et al., 2015), but these studies focused on frameshifts programmed into mRNA transcripts through combinations of four mechanisms: (1) use of rare codons to slow translation speed at the skip site, (2) weak base pairing of the P-site tRNA anticodon and mRNA codon, (3) strong base pairing of the P-site tRNA anticodon to the location where the ribosome will re-bind the mRNA, and (4) a region six bases upstream of the re-binding site that mimics a Shine-Dalgarno sequence and offsets the energetic cost of frameshifting (Pech et al., 2010). Although the UAG codon in our GFP transcript slows translation, the P-site codon-anticodon pair for the codon immediately upstream of UAG is exact (CAC codon and ^GUGHis-tRNA anticodon) (Hsu et al., 1984) and any frameshift except backward would incur greater mispairing between the P site codon and anticodon. Additionally, no Shine Dalgarno-like sequence (AGGAGG) (Shine and Dalgarno, 1974; Vimberg et al., 2007) exists upstream, suggesting that the GFP construct we use contains only one of the four elements required for programmed ribosomal frameshifting (Supplementary file 1). From our construct, we observed frameshifts of potentially up to −6 and +19 nucleotides in response to the unassigned UAG codon (Figure 2, Supplementary file 1 – Tables S1 and S2). Collectively, our work uncovers a wide variety of frameshifting events that can occur in response to ribosomal stalling in vivo, highlighting the capacity of the ribosome to continue translation despite missing an essential translational component.

Mass spectrometry analysis also revealed truncated mistranslation products that possibly represent loss of translational fidelity and termination by RF2 downstream of an initial mistranslation event at the UAG codon, known as post-peptidyl transfer quality control (Petropoulos et al., 2014; Zaher and Green, 2009), a result previously only observed in vitro. Although prior studies decades ago revealed premature truncation products in vivo (Manley, 1978), they lacked the technical capability to determine whether these peptides arose from a single mistranslation event or demonstrated loss of translational fidelity after the ribosome encounters a rare or unassigned codon. The mistranslation products we detect show repeated mistranslation events that could not have been produced by suppression, ribosomal rescue, or frameshifting, unless the ribosome frameshifted multiple times after resolving stalling at the UAG codon (Figure 2B, Supplementary file 1). These events may be followed by ribosomal rescue via ArfA or ArfB, spontaneous ribosomal dissociation, or termination via release factor 2, though our technique was not capable of distinguishing between these fates. Previous in vitro studies using purified ribosome complexes determined that a mistranslation event destabilized the P-site helix, reducing the ability of the A-site to discriminate between anticodons and resulting in further mistranslation events and rapid termination by RF2 with the assistance of release factor 3 (Zaher and Green, 2009; Zaher and Green, 2010). The researchers predicted that a single mistranslation event would also lead to prematurely truncated peptides with two or three miscoded C-terminal amino acids appended in vivo (Zaher and Green, 2009). These findings, together with our results, motivate future work to investigate the possibility of loss of translational fidelity after an initial translation error and highlight the GRO as a model for elucidating translational fidelity in vivo.

The GRO demonstrates that general ribosomal rescue mechanisms resolve ribosomal stalling at unassigned stop codons. As most sequenced bacterial species contain a homolog of the tmRNA, ArfA, or ArfB ribosomal rescue systems (Hudson et al., 2014; Keiler, 2015) and eukaryotic cells contain analogous pathways that rescue stalled ribosomes (Graille and Séraphin, 2012), we anticipate that translational stalling at unassigned codons can be resolved similarly in these organisms. Accordingly, we hypothesize that organisms beyond E. coli should tolerate unassigned codons as intermediates toward codon reassignments in genomic recoding, efforts for which are underway in numerous prokaryotic and eukaryotic species (Lau et al., 2017; Napolitano et al., 2016; Ostrov et al., 2016; Richardson et al., 2017). Additional barriers to codon reassignment exist, such as regulatory roles of codons in gene expression (Lajoie et al., 2013a), but our findings indicate that unassigned codons are tolerable in the absence of specialized translational machinery to address them, both as intermediate steps towards codon reassignment and as permanent parts of the genetic code.

Our findings suggest that we can use unassigned codons to engineer organisms with broad resistance to HTGEs and impart genetic isolation, increasing engineered organisms’ stability in biotechnology applications. Since tmRNA homologs are found in >99% of all sequenced bacterial genomes (Hudson et al., 2014; Keiler, 2015), we would expect other organisms engineered to contain unassigned codons to exhibit immunity to horizontally transferred genetic elements. As researchers pursue further efforts in whole genome recoding (Boeke et al., 2016; Lau et al., 2017; Napolitano et al., 2016; Ostrov et al., 2016; Richardson et al., 2017) and engineer organisms for use in open environments, we require strategies to genetically isolate such organisms from their surrounding environment to ensure robust function, both individually (Moe-Behrens et al., 2013) and as members of microbial communities (Grosskopf and Soyer, 2014; Hillesland and Stahl, 2010). Genomically recoded organisms with unassigned codons would possess reduced susceptibility to exploitation by HTGEs, increasing their stability in open environments. Although this work demonstrates that an unassigned stop codon acts as a barrier to HGT, this current barrier can be breached by mutation or deletion of the tmRNA to produce a functional protein. In contrast, we expect that an organism with an unassigned sense codon would have even greater barriers to HGT, as premature termination at an unassigned sense codon would likely produce a nonfunctional, truncated peptide. We thus anticipate that further genomic recoding to engineer additional unassigned sense and nonsense codons may be a broadly applicable strategy to confer genetic isolation in living systems, facilitating the safe use of engineered organisms in complex open environments.

Sequences of strains used have been previously published with the appropriate citations. Modifications (e.g., gene deletions) to those strains are described in full in the Tables, Key Resource Guide, methods and supplementary material. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD009643 (http://proteomecentral.proteomexchange.org) via the PRIDE partner repository (Vizcaíno et al, 2014).

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Author details

1. Natalie Jing Ma

   1. Department of Molecular, Cellular & Developmental Biology, Yale University, New Haven, United States
   2. Systems Biology Institute, Yale University, West Haven, United States

   Contribution

   Conceptualization, Formal analysis, Validation, Investigation, Visualization, Methodology, Writing—original draft, Writing—review and editing

   Contributed equally with

   Colin F Hemez and Karl W Barber

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0001-7452-9482

2. Colin F Hemez

   1. Systems Biology Institute, Yale University, West Haven, United States
   2. Department of Biomedical Engineering, Yale University, New Haven, United States

   Contribution

   Investigation, Visualization, Writing—original draft, Writing—review and editing

   Contributed equally with

   Natalie Jing Ma and Karl W Barber

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0003-3445-7706

3. Karl W Barber

   1. Systems Biology Institute, Yale University, West Haven, United States
   2. Department of Cellular & Molecular Physiology, Yale University School of Medicine, New Haven, United States

   Contribution

   Resources, Investigation, Visualization, Writing—review and editing

   Contributed equally with

   Natalie Jing Ma and Colin F Hemez

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0003-0672-8409

4. Jesse Rinehart

   1. Systems Biology Institute, Yale University, West Haven, United States
   2. Department of Cellular & Molecular Physiology, Yale University School of Medicine, New Haven, United States

   Contribution

   Resources, Supervision, Funding acquisition, Validation, Methodology

   For correspondence

   jesse.rinehart@yale.edu

   Competing interests

   No competing interests declared

5. Farren J Isaacs

   1. Department of Molecular, Cellular & Developmental Biology, Yale University, New Haven, United States
   2. Systems Biology Institute, Yale University, West Haven, United States

   Contribution

   Conceptualization, Supervision, Funding acquisition, Methodology, Writing—original draft, Writing—review and editing

   For correspondence

   farren.isaacs@yale.edu

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0001-8615-8236

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