Journal: PLoS Genetics
Article Title: Modification of tRNA Lys UUU by Elongator Is Essential for Efficient Translation of Stress mRNAs
doi: 10.1371/journal.pgen.1003647
Figure Lengend Snippet: (A and B) Vectors carrying a constitutively expressed wild-type (p HA-atf1 ′) or mutated atf1 gene (p HA-atf1 AAG ′) were integrated in the chromosomes of wild-type or Δsin3/elp3 strains. Rich media cultures of strains JF91 (WT+p HA-atf1 ′), JF92 ( Δsin3/elp3 +p HA-atf1 ′), JF94 (WT+p HA-atf1 AAG ′) and JF95 ( Δsin3/elp3 +p HA-atf1 AAG ′), either untreated (0) or treated with 1 mM H 2 O 2 for the indicated times, were analyzed to determine HA-atf1 mRNA levels by Northern blot using an anti-HA probe (A) or HA-Atf1 protein levels by Western blot using monoclonal antibody against HA (B). The numbers below the Northern or Western blot panels indicate the relative levels of HA-atf1/act1 mRNAs (panel A) or HA-Atf1/tubulin protein levels (panel B). (C) Expression of the mutant AAA-to-AAG Atf1 protein does not suppress the growth defects of Δsin3/elp3 cells upon oxidative stress. Empty vector or plasmids carrying a wild-type (p HA-atf1 ′) or a mutated atf1 gene (p HA-atf1 AAG ′) were integrated in the chromosomes of wild-type or Δsin3/elp3 strains. Cultures from the resulting strains JF88 (WT+empty vector), JF89 ( Δsin3/elp3 +empty vector), JF91 (WT+p HA-atf1 ′), JF92 ( Δsin3/elp3 +p HA-atf1 ′), JF94 (WT+p HA-atf1 AAG ′) and JF95 ( Δsin3/elp3 +p HA-atf1 AAG ′) were serially diluted and spotted onto rich media plates without (Untreated) or with 1 mM H 2 O 2 .
Article Snippet: To generate a mutated version of atf1 where all eleven AAA codons were replaced by AAG, full length gene synthesis was performed (GeneScript).
Techniques: Northern Blot, Western Blot, Expressing, Mutagenesis, Plasmid Preparation