Ribosomal Frameshifting Selectively Modulates the Assembly, Function, and Pharmacological Rescue of a Misfolded CFTR Variant.

The cotranslational misfolding of the cystic fibrosis transmembrane conductance regulator chloride channel (CFTR) plays a central role in the molecular basis of cystic fibrosis (CF). The misfolding of the most common CF variant (ΔF508) remodels both the translational regulation and quality control of CFTR. Nevertheless, it is unclear how the misassembly of the nascent polypeptide may directly influence the activity of the translation machinery. In this work, we identify a structural motif within the CFTR transcript that stimulates efficient -1 ribosomal frameshifting and triggers the premature termination of translation. Though this motif does not appear to impact the interactome of wild-type CFTR, silent mutations that disrupt this RNA structure alter the association of nascent ΔF508 CFTR with numerous translation and quality control proteins. Moreover, disrupting this RNA structure enhances the functional gating of the ΔF508 CFTR channel at the plasma membrane and its pharmacological rescue by the CFTR modulators contained in the CF drug Trikafta. The effects of the RNA structure on ΔF508 CFTR appear to be attenuated in the absence of the ER membrane protein complex (EMC), which was previously found to modulate ribosome collisions during preemptive quality control of a misfolded CFTR homolog. Together, our results reveal that ribosomal frameshifting selectively modulates the assembly, function, and pharmacological rescue of a misfolded CFTR variant. These findings suggest interactions between the nascent chain, quality control machinery, and ribosome may dynamically modulate ribosomal frameshifting in order to tune the processivity of translation in response to cotranslational misfolding.

General trends in the effects of VX-661 and VX-445 on the plasma membrane expression of clinical CFTR variants.

Cystic fibrosis (CF) is caused by mutations that compromise the expression and/or function of the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel. Most people with CF harbor a common misfolded variant (ΔF508) that can be partially rescued by therapeutic "correctors" that restore its expression. Nevertheless, many other CF variants are insensitive to correctors. Using deep mutational scanning, we quantitatively compare the effects of two correctors on the plasma membrane expression of 129 CF variants. Though structural calculations suggest corrector binding provides similar stabilization to most variants, it's those with intermediate expression and mutations near corrector binding pockets that exhibit the greatest response. Deviations in sensitivity appear to depend on the degree of variant destabilization and the timing of misassembly. Combining correctors appears to rescue more variants by doubling the binding energy and stabilizing distinct cotranslational folding transitions. These results provide an overview of rare CF variant expression and establish new tools for precision pharmacology.

Synthetic auxotrophy remains stable after continuous evolution and in coculture with mammalian cells.

Understanding the evolutionary stability and possible context dependence of biological containment techniques is critical as engineered microbes are increasingly under consideration for applications beyond biomanufacturing. While synthetic auxotrophy previously prevented Escherichia coli from exhibiting detectable escape from batch cultures, its long-term effectiveness is unknown. Here, we report automated continuous evolution of a synthetic auxotroph while supplying a decreasing concentration of essential biphenylalanine (BipA). After 100 days of evolution, triplicate populations exhibit no observable escape and exhibit normal growth rates at 10-fold lower BipA concentration than the ancestral synthetic auxotroph. Allelic reconstruction reveals the contribution of three genes to increased fitness at low BipA concentrations. Based on its evolutionary stability, we introduce the progenitor strain directly to mammalian cell culture and observe containment of bacteria without detrimental effects on HEK293T cells. Overall, our findings reveal that synthetic auxotrophy is effective on time scales and in contexts that enable diverse applications.

Release Factor Inhibiting Antimicrobial Peptides Improve Nonstandard Amino Acid Incorporation in Wild-type Bacterial Cells.

We report a tunable chemical genetics approach for enhancing genetic code expansion in different wild-type bacterial strains that employ apidaecin-like, antimicrobial peptides observed to temporarily sequester and thereby inhibit Release Factor 1 (RF1). In a concentration-dependent matter, these peptides granted a conditional lambda phage resistance to a recoded Escherichia coli strain with nonessential RF1 activity and promoted multisite nonstandard amino acid (nsAA) incorporation at in-frame amber stop codons in vivo and in vitro. When exogenously added, the peptides stimulated specific nsAA incorporation in a variety of sensitive, wild-type (RF1+) strains, including Agrobacterium tumefaciens, a species in which nsAA incorporation has not been previously reported. Improvement in nsAA incorporation was typically 2-15-fold in E. coli BL21, MG1655, and DH10B strains and A. tumefaciens with the >20-fold improvement observed in probiotic E. coli Nissle 1917. In-cell expression of these peptides promoted multisite nsAA incorporation in transcripts with up to 6 amber codons, with a >35-fold increase in BL21 showing moderate toxicity. Leveraging this RF1 sensitivity allowed multiplexed partial recoding of MG1655 and DH10B that rapidly resulted in resistant strains that showed an additional approximately twofold boost to nsAA incorporation independent of the peptide. Finally, in-cell expression of an apidaecin-like peptide library allowed the discovery of new peptide variants with reduced toxicity that still improved multisite nsAA incorporation >25-fold. In parallel to genetic reprogramming efforts, these new approaches can facilitate genetic code expansion technologies in a variety of wild-type bacterial strains.