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. 2017 Jun 15;37(13):e00029-17. doi: 10.1128/MCB.00029-17

FIG 5.

FIG 5

Cet1's NTD recruits the Paf1C via FACT to promote transcription. (A and B) Cet1's NTD enhances recruitment of the Paf1C to the promoter-proximal sites (or 5′ ORFs) of ADH1, PGK1, PMA1, and PYK1. (C) Analysis of Paf1 and actin levels in strains expressing PAF1 under the control of the GAL1 promoter (PGAL1-PAF1-HA) after switching the carbon source in the growth medium from galactose (when total OD600 was 0.6) to dextrose. (D and E) Analysis of Rpb1 association with the ADH1, PGK1, PYK1, and PMA1 coding sequences in the strains expressing PAF1 under the control of the GAL1 promoter (PGAL1-PAF1-HA) after switching the carbon source in the growth medium from galactose to dextrose. (F) RT-PCR analysis of ADH1, PGK1, PYK1, and PMA1 mRNAs in strains expressing PAF1 under the control of the GAL1 promoter (PGAL1-PAF1-HA) after switching the carbon source in the growth medium from galactose to dextrose. (G) Co-IP analysis between Cet1 and Paf1 without in vivo cross-linking by formaldehyde. (H) Analysis of Pob3 and Paf1 levels in strains expressing HA-tagged Pob3 under the control of the GAL1 promoter (PGAL1-POB3-HA) after switching the carbon source in the growth medium from galactose to dextrose. (I) FACT enhances the recruitment of Paf1C to the promoter-proximal sites (or 5′ ORFs) of ADH1, PGK1, PMA1, and PYK1. (J and K) Pob3 is required for recruitment of Spt16 to the active gene. (J) Levels of Pob3 and Spt16 (by Western blot analysis) in the strain expressing HA-tagged Pob3 under the control of the GAL1 promoter (PGAL1-POB3-HA) after switching the carbon source in the growth medium from galactose to dextrose. (K) ChIP analysis of Spt16 and Pob3 at the ADH1 coding sequence following shutdown of Pob3 expression in dextrose-containing growth medium for 4 h. The error bars indicate SD.