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a. Schematic representation of two transcriptional phenotypes used in this study. Mycophenolic acid (MPA) sensitivity: MPA inhibits the yeast inosine monophosphate dehydrogenase (IMPDH) enzymes encoded by IMD3 and IMD4 , leading to GTP depletion. In response, wild-type cells induce IMD2 expression by a TSS shift from an upstream “G” TSSs, which produces nonfunctional cryptic transcripts, to a downstream “A” TSS that yields a functional IMD2 mRNA. Mutants that are unable to shift the usage to the downstream TSS show MPA S (top). Suppression of imd2 Δ ::HIS3 : The IMD2 ORF was replaced with the HIS3 to generate the reporter construct. In the absence of MPA, wild-type cells initiate <t>transcription</t> from upstream TSSs of the IMD2 promoter, producing nonfunctional transcripts and failing to grow on medium lacking histidine (SC-His). Mutants that shift transcription constitutively to the downstream TSS express HIS3 and show a His⁺ phenotype (bottom). b. Schematic of genetic screening by PCR-based random mutagenesis coupled with gap repair/plasmid shuffle to examine tfb3 or tfa1 alleles. c. Initiation phenotypes of select representative novel tfb3 mutants obtained from genetic screening. d. Initiation phenotypes of select representative novel tfa1 mutants obtained from genetic screening. 10-fold serial dilutions of saturated cultures of WT and mutant strains were plated on different phenotyping media. e. Positions of Tfb3 and Tfa1 relative to TFIIH and Pol II in the yeast PIC (PDB 7O4J) ( S chilbach et al . 2021 ). f. Mapping novel tfb3 and tfa1 mutant residues shown as spheres on the cartoon representation of Tfb3 and Tfa1 protein structures from ( e ). Residues are colored according to phenotype: shades of purple indicate increasing MPA S mutant strength; shades of orange indicate increasing His⁺ mutant strength; exhibiting with distinct substitutions conferring MPA S or His⁺ phenotypes are shown in red.
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a. Schematic representation of two transcriptional phenotypes used in this study. Mycophenolic acid (MPA) sensitivity: MPA inhibits the yeast inosine monophosphate dehydrogenase (IMPDH) enzymes encoded by IMD3 and IMD4 , leading to GTP depletion. In response, wild-type cells induce IMD2 expression by a TSS shift from an upstream “G” TSSs, which produces nonfunctional cryptic transcripts, to a downstream “A” TSS that yields a functional IMD2 mRNA. Mutants that are unable to shift the usage to the downstream TSS show MPA S (top). Suppression of imd2 Δ ::HIS3 : The IMD2 ORF was replaced with the HIS3 to generate the reporter construct. In the absence of MPA, wild-type cells initiate transcription from upstream TSSs of the IMD2 promoter, producing nonfunctional transcripts and failing to grow on medium lacking histidine (SC-His). Mutants that shift transcription constitutively to the downstream TSS express HIS3 and show a His⁺ phenotype (bottom). b. Schematic of genetic screening by PCR-based random mutagenesis coupled with gap repair/plasmid shuffle to examine tfb3 or tfa1 alleles. c. Initiation phenotypes of select representative novel tfb3 mutants obtained from genetic screening. d. Initiation phenotypes of select representative novel tfa1 mutants obtained from genetic screening. 10-fold serial dilutions of saturated cultures of WT and mutant strains were plated on different phenotyping media. e. Positions of Tfb3 and Tfa1 relative to TFIIH and Pol II in the yeast PIC (PDB 7O4J) ( S chilbach et al . 2021 ). f. Mapping novel tfb3 and tfa1 mutant residues shown as spheres on the cartoon representation of Tfb3 and Tfa1 protein structures from ( e ). Residues are colored according to phenotype: shades of purple indicate increasing MPA S mutant strength; shades of orange indicate increasing His⁺ mutant strength; exhibiting with distinct substitutions conferring MPA S or His⁺ phenotypes are shown in red.

Journal: bioRxiv

Article Title: RNA polymerase II-TFIIE-TFIIH interface functions in transcription start site selection in Saccharomyces cerevisiae

doi: 10.1101/2025.11.05.686797

Figure Lengend Snippet: a. Schematic representation of two transcriptional phenotypes used in this study. Mycophenolic acid (MPA) sensitivity: MPA inhibits the yeast inosine monophosphate dehydrogenase (IMPDH) enzymes encoded by IMD3 and IMD4 , leading to GTP depletion. In response, wild-type cells induce IMD2 expression by a TSS shift from an upstream “G” TSSs, which produces nonfunctional cryptic transcripts, to a downstream “A” TSS that yields a functional IMD2 mRNA. Mutants that are unable to shift the usage to the downstream TSS show MPA S (top). Suppression of imd2 Δ ::HIS3 : The IMD2 ORF was replaced with the HIS3 to generate the reporter construct. In the absence of MPA, wild-type cells initiate transcription from upstream TSSs of the IMD2 promoter, producing nonfunctional transcripts and failing to grow on medium lacking histidine (SC-His). Mutants that shift transcription constitutively to the downstream TSS express HIS3 and show a His⁺ phenotype (bottom). b. Schematic of genetic screening by PCR-based random mutagenesis coupled with gap repair/plasmid shuffle to examine tfb3 or tfa1 alleles. c. Initiation phenotypes of select representative novel tfb3 mutants obtained from genetic screening. d. Initiation phenotypes of select representative novel tfa1 mutants obtained from genetic screening. 10-fold serial dilutions of saturated cultures of WT and mutant strains were plated on different phenotyping media. e. Positions of Tfb3 and Tfa1 relative to TFIIH and Pol II in the yeast PIC (PDB 7O4J) ( S chilbach et al . 2021 ). f. Mapping novel tfb3 and tfa1 mutant residues shown as spheres on the cartoon representation of Tfb3 and Tfa1 protein structures from ( e ). Residues are colored according to phenotype: shades of purple indicate increasing MPA S mutant strength; shades of orange indicate increasing His⁺ mutant strength; exhibiting with distinct substitutions conferring MPA S or His⁺ phenotypes are shown in red.

Article Snippet: A reverse transcription reaction was performed by mixing M-MuLV Reverse Transcriptase (NEB, RNase inhibitor (NEB), dNTPs (GE), DTT, and labeled primer with total RNA.

Techniques: Expressing, Functional Assay, Construct, Mutagenesis, Plasmid Preparation