(a) Five forms of cytosine residues. 5mC modification is catalyzed by DNA methyltransferase. Oxidation of 5mC by TET proteins produces 5hmC, 5fC, and 5caC, respectively. (b) Transcriptional elongation scaffolds for NTP incorporation on modified cytosine templates (denoted by X). RNA, template strand DNA (TS) and nontemplate strand DNA (NTS) are shown in red, cyan, and green, respectively. Modified cytosine residues are highlighted in orange. The 3’-ends of RNA residues are underlined. (c) Relative NTP incorporation efficiency on all five modified cytosine templates by mammalian Pol II (scaffold A). Data from C, 5mC, 5hmC, 5fC, and 5caC templates were shown in blue, magenta, cyan, red and orange, respectively. Data were normalized to GTP incorporation efficiency for C template at 15 sec. All error bars (standard deviation) are derived from three experiments. (d) Template-dependent NTP incorporation by yeast Pol II (scaffold A) and relative NTP incorporation efficiency on all five modified cytosine templates. Data were normalized to GTP incorporation efficiency for C template at 15 sec. All error bars (standard deviation) are derived from four experiments. Color codes are the same as (c). (e) Template-dependent NTP incorporation by yeast Pol II (scaffold B) and relative Pol II transcription efficiency on modified cytosine templates. Data were normalized to product formation for C template at the same experimental condition. All error bars (standard deviation) are derived from three experiments. Color codes are the same as (c). The band position corresponding to GTP incorporation opposite to modification site was depicted with an arrow in the gel.

(a) Pol II substrate specificity of GTP and ATP incorporation for C and 5fC templates respectively. (b) Pol II substrate discrimination of GTP over ATP incorporation for C and 5fC templates. Data from C and 5fC templates are shown in blue and red, respectively. All error bars (standard deviation) are derived from three experiments.

(a) Pol II polymerization rates for GTP incorporation on C, 5hmC, 5fC, and 5caC templates. (b) Relative Pol II substrate specificity of GTP incorporation for C, 5hmC, 5fC, and 5caC templates. The Pol II specificity (k_pol/K_d,app) was normalized to that for C template. All error bars (standard deviation) are derived from three experiments. (c) Rates of RNA cleavage stimulated by Transcription Factor IIS for C, 5mC, 5hmC, 5fC, and 5caC templates. Error bars represent deviations in non-linear regression analysis. Color codes are the same as Fig. 1c.