Project description:Transfer RNAs (tRNAs) maintain translational fidelity through strict charging by their cognate aminoacyl-tRNA synthetase and codon:anticodon base pairing with the mRNA at the ribosome. Mistranslation occurs when an amino acid not specified by the genetic code is incorporated into a protein. Since alanyl-tRNA synthetase uniquely recognizes a G3:U70 base pair in alanine tRNAs and the anticodon plays no role in charging, alanine tRNA variants with anticodon mutations have the potential to mistranslate alanine. Our goal was to quantify mis-incorporation of alanine into proteins in Saccharomyces cerevisiae strains expressing one of 57 different alanine tRNA anticodon variants. Using mass spectrometry, we observed mistranslation for 45 of the variants when expressed on single-copy plasmids.
Project description:A tet-off strain of Saccharomyces cerevisiae was constructed in which the GLN4 glutamine tRNA synthetase gene was placed under control of a doxycycline-regulated promoter. The transcriptional responses to Gln4p tRNA synthetase depletion were assessed by growth of the strain in the presence, or absence, of doxycycline (1 µg/ml). A control, wild-type strain was similarly treated with doxycycline or left untreated as a reference. Each strain/condition RNA isolation was performed using triplicate independent biological samples A, B and C.
Project description:Transcription profiles of mutant M1 and the control in the absence/presence of 4mM H2O2 M1 is a mutated Saccharomyces cerevisiae strain. It was obtained through transcriptional engineering of general transcription factor TFIIB. Encoded by SUA7, TFIIB is reported to regulate the expression of over 730 genes in Saccharomyces cerevisiae. M1 showed significant growth improvement compared with the control when challenged with 4mM H2O2. It also had resistance towards high osmotic pressure (1.5M NaCl).
Project description:We used genetic code expansion technology with an engineered Saccharomyces cerevisiae tryptophanyl tRNA- synthetase:suppressor tRNA pair in Escherichia coli, to directly insert the non- canonical amino acid 5-OH tryptophan (5-OHTrp) at position 72 in human apoA-I. Characterization of recombinant human apoA-I by mass spectrometry confirmed successful site-specific incorporation of Trp(5-OH) in apoA-I.
Project description:Here we report the design, construction and characterization of a tRNA neochromosome, a designer chromosome that functions as an additional, de novo counterpart to the native complement of Saccharomyces cerevisiae chromosomes. Intending to address one of the central design principles of the Sc2.0 project, the ~190 kb tRNA neochromosome houses all 275 relocated nuclear tRNA genes. To maximize stability, the design incorporated orthogonal genetic elements from non-S. cerevisiae yeast species. Furthermore, the presence of 283 rox recombination sites enable an orthogonal SCRaMbLE system capable of adjusting tRNA abundance. Following construction, we obtained evidence of a potent selective force once the neochromosome was introduced into yeast cells, manifesting as a spontaneous doubling in cell ploidy. Furthermore, tRNA sequencing, transcriptomics, proteomics, nucleosome mapping, replication profiling, FISH and Hi-C were undertaken to investigate questions of tRNA neochromosome behavior and function. Its construction demonstrates the remarkable tractability of the yeast model and opens up new opportunities to directly test hypotheses surrounding these essential non-coding RNAs.
