(A) Intracellular pools of amino acids are increased in tRNA thiolation mutants. Steady-state amino acid amounts were measured in wild-type (WT) and tRNA thiolation mutant cells (uba4Δ and ncs2Δ) …
(A) GCN4 translation is increased in tRNA thiolation mutants. A schematic representation of different Gcn4-luciferase (Gcn4-luc) translational reporter constructs. Two upstream ORFs in the 5’ UTR of …
(A) Intracellular levels of nucleotides are decreased in tRNA thiolation mutants. Steady-state nucleotide (AMP, GMP, CMP and UMP) amounts were measured in wild-type (WT) and tRNA thiolation mutant (u…
(A) Schematic representation depicting nucleotide and storage carbohydrates (trehalose and glycogen) biosynthesis, starting from a common precursor, glucose-6-phosphate. In normal conditions where …
(A) Nucleotide synthesis is decreased in tRNA thiolation mutants. Wild-type (WT) and tRNA thiolation mutant cells (ncs2Δ) grown in minimal media were pulse-labelled with [U-13C6]-labelled glucose …
(A) A schematic representation illustrating the experimental design for sulfur starvation. Wild type cells growing in standard glucose medium were shifted to sulfur-rich and sulfur-starved/sulfur …
(A) tRNA thiolation mutants exhibit asynchronous cell division and disrupted yeast metabolic cycles. WT and tRNA thiolation mutant cells (uba4Δ) growing in chemostat cultures under conditions of …
(A) Catalytically dead uba4 mutants (C225A and C397A) exhibit increased HU sensitivity. Wild-type (WT), tRNA thiolation mutant cells (uba4Δ and ncs2Δ) and catalytically dead uba4 mutant (C225A and …
(A) Correlation plots are shown, for WT and tRNA thiolation mutant cells (ncs2Δ and uba4Δ), for both gene expression (transcript) and ribosome footprint (translation) changes. The coefficients of …
(A) Correlation plots, for RNA (transcript) or ribosome footprint (RPF), between biological replicates, of WT, or thiolation mutant cells. Note, correlation coefficients (R) for any replicate of the …
(A) Changes in the ribosome density based on codon occupancy are shown at A, P, and E sites. Different codons are highlighted in both plots. n = 3 biological replicates for each strain are plotted. …
(A) Heat maps representing changes in expression (transcript and ribosome footprints) of genes involved in nucleotide biosynthesis, for wild-type (WT) and tRNA thiolation mutant cells (uba4Δ and ncs2…
(A) A schematic representation of PHO regulon-related genes, and their roles. Pho84 and Pho89 are high affinity phosphate transporters, Pho87 and Pho90 are low affinity phosphate transporters, Spl2 …
(A) Pho12 protein is decreased in tRNA thiolation mutants in high and low Pi conditions. Using cells expressing endogenous Pho12 with a FLAG epitope at its carboxy-terminus, Pho12 protein levels in …
(A) Trehalose amounts are increased upon phosphate starvation. Trehalose content of WT cells grown in high and no Pi media was plotted. Data are displayed as means ± SD, n = 3. ****p<0.0001. (B) …
(A) GCN4 translation is Gcn2-dependent in phosphate-starved wild-type cells. Wild-type (WT) and gcn2Δ cells transformed with the WT Gcn4-luc construct were grown in high and no phosphate medium …
(A) A schematic representation of the trehalose cycle, showing the routing of glucose-6-phospate towards trehalose and glycogen biosynthesis. Trehalose synthesis requires two glucose molecules, …
(A) Intracellular Pi levels are maintained in tRNA thiolation mutant cells (uba4Δ and ncs2Δ) by Tps2 activity. Free intracellular Pi levels were determined in wild-type (WT), tRNA thiolation mutants …
WT cells (which have a functional tRNA thiolation machinery) in amino acid (methionine and cysteine) replete conditions have high amounts of thiolated tRNAs. In these cells, carbon flux coupled with …
Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
---|---|---|---|---|
Antibody | anti-HA | Roche | Cat# 12CA5 | WB (1:2000) |
Antibody | anti-phospho eIF2a (Ser51) | Cell Signalling Technology | Cat# 9721S | WB (1:1000) |
Antibody | anti-FLAG | Sigma-Aldrich | Cat # F1804-5MG | WB (1:2000) |
Antibody | anti-Rad53 yC-19 | Santa Cruz Biotechnology | Cat # sc-6749 | WB (>1:1000) |
Antibody | HRP-conjugated secondary antibodies (anti-mouse, anti-rabbit) | Sigma-Aldrich | Cat # 7076S Cat # 7074S | WB (1:5000) |
HPLC column | Synergi 4µ Fusion-RP 80A column (100 × 4.6 mm) | Phenomenex | Cat # 00D-4424-E0 | |
Sequence-based reagent | Gcn4-luciferase translation reporters | this study | ||
Peptide, recombinant protein | trehalase | Sigma-Aldrich | Cat # T8778 | |
Peptide, recombinant protein | amyloglucosidase | Sigma-Aldrich | Cat # 10115 | |
Peptide, recombinant protein | RNAse A | Sigma-Aldrich | Cat # R4875 | |
Peptide, recombinant protein | protease solution | Sigma-Aldrich | Cat # P6887 | |
Commercial assay or kit | Glucose estimation kit | Sigma-Aldrich | Cat # GAGO20 | |
Commercial assay or kit | RiboZero | Epicenter | Cat # MRZH116 | |
Commercial assay or kit | luciferase assay kit | Promega | Cat # E1500 | |
Commercial assay or kit | Maxima SYBRGreen/ROX qPCR Master Mix | Thermo Scientific | Cat # K0222 | |
Commercial assay or kit | ATP estimation kit | Thermo Scientific | Cat # A22066 | |
Chemical compound, drug | SYTOX green | Thermo Scientific (Invitrogen) | Cat # S7020 | |
Chemical compound, drug | p-nitrophenyl phosphate | Sigma-Aldrich | Cat # N4645 | |
Chemical compound, drug | bicinchoninic acid assay | Thermo Scientific | Cat # 23225 | |
Software, algorithm | Prism 7 | Graphpad |
Strains used in this study.
Plasmids used in this study.
Mass transitions for detection of metabolites.
RNA-RFP changes (mutant-WT).