Repurposing tRNAs for nonsense suppression

Abstract

Three stop codons (UAA, UAG and UGA) terminate protein synthesis and are almost exclusively recognized by release factors. Here, we design de novo transfer RNAs (tRNAs) that efficiently decode UGA stop codons in Escherichia coli. The tRNA designs harness various functionally conserved aspects of sense-codon decoding tRNAs. Optimization within the TΨC-stem to stabilize binding to the elongation factor, displays the most potent effect in enhancing suppression activity. We determine the structure of the ribosome in a complex with the designed tRNA bound to a UGA stop codon in the A site at 2.9 Å resolution. In the context of the suppressor tRNA, the conformation of the UGA codon resembles that of a sense-codon rather than when canonical translation termination release factors are bound, suggesting conformational flexibility of the stop codons dependent on the nature of the A-site ligand. The systematic analysis, combined with structural insights, provides a rationale for targeted repurposing of tRNAs to correct devastating nonsense mutations that introduce a premature stop codon.

Genome-wide screening reveals metabolic regulation of translational fidelity

“…Random mutagenesis of rpsL and rRNA genes has later been performed to identify mutations that affect ribosomal fidelity (24,54,55). More recently, suppression of stop codons has attracted tremendous interest in the treatment of genetic diseases caused by nonsense mutations (56)(57)(58)(59)(60)(61). Several studies have used luminescence-based assays to screen for small compounds that promote the readthrough of stop codons (61,62).…”

Anticodon sequence determines the impact of mistranslating tRNAAlavariants

“…These tRNA Ala anticodons variants likely have the potential to misread stop codons since many stop codon suppressors are tRNAs with altered anticodons (80). The minimal impact of these variants likely results from competition with abundant and efficient translation release factors (81).…”

A tRNA Modification-based strategy for Identifying amiNo acid Overproducers (AMINO)

“…For , a modified tRNA with G73 mutation that led to lowered affinity to AsnRS could be used to construct a whole-cell-based selection system as reported 18,74 . Furthermore, other codon/anticodon pairs such as UGA/tRNA UCA , UCG/tRNA CGA , and UCA/tRNA UGA are alternatives to our selection system 72,75,76 .CUA…”

“…Modification of tRNA anticodon decreased the aminoacylation rate in varying degrees, thereby resulting in different selection stringency (Supplementary Table 1) when the tRNA CUA -based system was applied to different amino acids. Other engineered tRNA CUA s, nonsense codons, and corresponding decoding tRNAs, as well as orthogonal aaRS-tRNA pairs, are alternatives for adjusting the efficiency of the selection system 54,76,79 .…”