FIG 4.

Effect of high or low abundance of Spt16 on the engagement of elongating RNA polymerase II in transcription. (A) Spt16 expression analysis under the control of GAL1 promoter (P_GAL1-SPT16) in galactose-containing growth medium (or YPG) by Western blotting assay. Yeast cells were initially grown at 30°C to an OD₆₀₀ of 0.6 and were then collected immediately (0 h) and after 4 h. (B and C) ChIP analysis of Rpb1 association with ADH1, PGK1, PMA1, and PYK1 (ORF2 [B] and core promoter [C]) following overexpression of Spt16 in YPG. (D) RT-PCR analysis of ADH1, PGK1, PMA1, and PYK1 mRNAs following overexpression of Spt16 in YPG. (E) Growth analysis of the yeast strains expressing SPT16 under the control of the GAL1 promoter (P_GAL1-SPT16) or of its own endogenous promoter (WT) in the solid SC-uracil medium containing 2% galactose with (top panel) or without (bottom panel) 6-AU (100 μg/ml) at 30°C. (F) Analysis of Spt16 expression under the control of GAL1 promoter in dextrose-containing growth medium (or YPD) by Western blotting assay. (G and H) ChIP analysis of Rpb1 association with the coding sequences (ORF2 [G]) and promoters (H) of the ADH1, PGK1, PMA1, and PYK1 genes following underexpression of Spt16 in YPD. (I) RT-PCR analysis of ADH1, PGK1, PMA1, and PYK1 mRNAs following underexpression of Spt16 in YPD.