Abstract
While translational read-through of stop codons by suppressor tRNAs is common in many bacteria, archaea and eukaryotes, this phenomenon has not yet been observed in the α-proteobacterium Caulobacter crescentus. Based on a previous report that C. crescentus and Escherichia coli tRNAHis have distinctive identity elements, we constructed E. coli tRNAHis CUA, a UAG suppressor tRNA for C. crescentus. By examining the expression of three UAG codon- containing reporter genes (encoding a β-lactamase, the fluorescent mCherry protein, or the C. crescentus xylonate dehydratase), we demonstrated that the E. coli histidyl-tRNA synthetase/tRNAHis CUA pair enables in vivo UAG suppression in C. crescentus. E. coli histidyl-tRNA synthetase (HisRS) or tRNAHis CUA alone did not achieve suppression; this indicates that the E. coli HisRS/tRNAHis CUA pair is orthogonal in C. crescentus. These results illustrate that UAG suppression can be achieved in C. crescentus with an orthogonal aminoacyl-tRNA synthetase/suppressor tRNA pair.
Highlights
Nonsense suppression has been observed in many biological systems
Since E. coli tRNAHis should be produced by correct processing of the precursor transcripts in C. crescentus, we constructed another transcription unit, tRNAHis2CUA, by replacing C. crescentus mature tRNAHis from the precursor transcript of tRNAHis with E. coli mature tRNAHis
The ffs promoter, which is derived from the constitutive promoter of the 4.5S RNA gene in C. crescentus [16], was inserted before the tRNAHis genes
Summary
Nonsense suppression (read-through of the stop codons UAG, UAA, or UGA) has been observed in many biological systems. Since nonsense mutations cause premature termination of translation, suppression of nonsense codons has been extensively applied in prokaryotes and lower eukaryotes for identifying gene functions by conditional expression of such genes [1,2]. Genetic studies showed that nonsense suppression is often mediated by suppressor tRNAs with altered anticodons that recognize nonsense codons and insert an amino acid in response. Such suppressors have been found in many organisms including E. coli [5], yeast [2], C. elegans [6,7], and human [8]
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Disclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.