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. 2013 Nov;33(21):4198–4211. doi: 10.1128/MCB.00525-13

Fig 6.

Fig 6

Mimicking CK2 phosphorylation of Spt2 leads to spurious transcription. (A) Diagram of the pGAL1::FLO8::HIS3 reporter construct. (B and C) The RI/II spt2 acidic mutant is not able to inhibit spurious transcription from the cryptic promoter of the pGAL1::FLO8::HIS3 construct. Below are serial dilutions of cultures of an spt2Δ (B) or spt2Δ cdc73Δ (C) strain containing the pGAL1::FLO8::HIS3 reporter and transformed with a plasmid carrying either the wild type or a mutated version of SPT2, as indicated. The cells were spotted onto synthetic complete medium lacking uracil (SC−URA) or lacking both uracil and histidine (SC−URA−HIS) and containing galactose (B) or glucose (C) as the carbon source. The photographs were taken after 1 or 2 days of incubation at 30°C. (D) The RI/II spt2 acidic mutant is not able to suppress spurious transcription from the cryptic promoter of the FLO8 gene. The cdc73Δ spt2Δ strain transformed with a plasmid carrying either the wild type or a mutated version of SPT2, as indicated, was grown in yeast extract-peptone-dextrose at 30°C and then shifted for the indicated times to 39°C. Total RNA was extracted and analyzed by Northern blotting with a probe for FLO8. SCR1 served as a loading control. The FLO8 probe identifies the full-length FLO8 mRNA and the FLO8 short RNA, which was shown previously to initiate from a cryptic promoter (34). (E) Mimicking permanent Spt2 phosphorylation does not affect Spt2 function at the SRG1/SER3 locus. The diagram explains the complex regulation of SER3 by the intergenic transcription of the noncoding DNA of SRG1. In wild-type cells, noncoding DNA of SRG1 is actively transcribed, and nucleosomes are refolded in the wake of transcription, resulting in the repression of SER3. In the absence of Spt2, nucleosomes are not refolded at noncoding DNA of SRG1, and SER3 is derepressed. (F) Wild-type (WT) or spt2Δ strains were transformed with an empty plasmid, a plasmid containing wild-type SPT2 (WT-SPT2), or nonphosphorylatable or acidic spt2 mutants. These cells were grown in yeast extract-peptone-dextrose at 30°C. Total RNA was extracted and analyzed by Northern blotting with a probe against SER3 and SCR1.