Supplemental Material for Berg et al.,2018

Supplemental Information contains Table S1 with strain information, Table S2 with oligonucleotide sequences, Figure S1 with sequence of synthetic DNA fragments, Figure S2 with <i>S. cereivisae</i> PIKK alignments, Figure S3 with half life of WT Tra1, Figure S4 with Tra1pr-LacZ data, Figure S5 with synthetic growth interactions between <i>tra1_Q3 </i>and various SAGA and NuA4 deletions, Figure S6 with mapping of mutated residues on Tra1 structure, Figure S7 with fractionation data, Figure S8 with Spt7 localization data, Figure S9 with western blot of Spt7, Figure S10 with coverage map of Spt20 from mass spectrometry data, Figure S11 with tagged Spt20 spot plate and Figure S12 with half life of various <i>tra1</i>_Q3suppressors. Tra1Q3 Spt20 Mass Spectrometry Data file contains peaks identified from mass spectrometry of Spt20 isolated from TAP-Ada2 pull downs of SAGA

C-terminal processing of yeast Spt7 occurs in the absence of functional SAGA complex-3

<p><b>Copyright information:</b></p><p>Taken from "C-terminal processing of yeast Spt7 occurs in the absence of functional SAGA complex"</p><p>http://www.biomedcentral.com/1471-2091/8/16</p><p>BMC Biochemistry 2007;8():16-16.</p><p>Published online 8 Aug 2007</p><p>PMCID:PMC1976419.</p><p></p>:5 into YP media containing 2% glucose (lane 2) or 2%galactose (lane 3) and grown for 30 minutes. Extracts were prepared by glass bead disruption and 100 μg separated by SDS-PAGE (5%). Flag-Spt7 was detected by Western blotting with anti-Flag antibody. Lane one contains 100 μg of protein extract prepared after growth of BY4741 in galactose-containing media. . CY1811 containing was grown in raffinose. Expression of Spt7 was induced by the addition of galactose then inhibited by the addition of YP media containing 2% glucose. Cells were grown for 90 minutes which was empirically determined as time 0. Additional equal volume samples were taken at time 0, 30, 60, 120 and 240 minutes (lanes 2–7). Protein extracts were prepared and equal volumes separated by SDS-Page (5%). Flag-Spt7 was detected by Western blotting (top panel) or stained with Coomassie Brilliant Blue (bottom panel)

Mistranslating the genetic code with leucine in yeast and mammalian cells

Translation fidelity relies on accurate aminoacylation of transfer RNAs (tRNAs) by aminoacyl-tRNA synthetases (AARSs). AARSs specific for alanine (Ala), leucine (Leu), serine, and pyrrolysine do not recognize the anticodon bases. Single nucleotide anticodon variants in their cognate tRNAs can lead to mistranslation. Human genomes include both rare and more common mistranslating tRNA variants. We investigated three rare human tRNALeu variants that mis-incorporate Leu at phenylalanine or tryptophan codons. Expression of each tRNALeu anticodon variant in neuroblastoma cells caused defects in fluorescent protein production without significantly increased cytotoxicity under normal conditions or in the context of proteasome inhibition. Using tRNA sequencing and mass spectrometry we confirmed that each tRNALeu variant was expressed and generated mistranslation with Leu. To probe the flexibility of the entire genetic code towards Leu mis-incorporation, we created 64 yeast strains to express all possible tRNALeu anticodon variants in a doxycycline-inducible system. While some variants showed mild or no growth defects, many anticodon variants, enriched with G/C at positions 35 and 36, including those replacing Leu for proline, arginine, alanine, or glycine, caused dramatic reductions in growth. Differential phenotypic defects were observed for tRNALeu mutants with synonymous anticodons and for different tRNALeu isoacceptors with the same anticodon. A comparison to tRNAAla anticodon variants demonstrates that Ala mis-incorporation is more tolerable than Leu at nearly every codon. The data show that the nature of the amino acid substitution, the tRNA gene, and the anticodon are each important factors that influence the ability of cells to tolerate mistranslating tRNAs.</p