Figure 7

TgCdk7 inhibition affects de novo transcription by TgRPB1.

(A) Schematic diagram showing approximate positions in Kb of the ChIP-qPCR products relative to the TUB1 and SAG1 start codons (upper panel). Occupancy of unphosphorylated TgRPB1 and P-Ser5 at the TUB1 and SAG1 promoters was determined using ChIP analysis. Immunoprecipitated DNA from inhibitor treated parasites was quantified and normalized by calculating fold enrichment over nontranscribed telomere DNA⁶², followed by real-time PCR. Prominent reduction of Ser5 phosphorylation was observed on TUB1 and ACT1 promoters at both the concentrations of inhibitor used with nearly two fold reductions at 10 μM of BS-181. However, unphosphorylated TgRPB1 occupancy at these promoters remains unchanged. (B) Schematic diagram showing approximate locations in Kb of the forward and reverse primers of qRT-PCR products relative to the TUB1 and ACT1 start codons (upper panel). Quantitative RT-PCR of the pre-mRNA levels of TUB1 and ACT1 was undertaken. A primer set targeting a region of ACT1 corresponding to exon1 and 2 was used as the control. Noticeable reduction was observed in the pre-mRNA levels of both the genes at 5 μM and 10 μM of BS-181. (C) Marked reduction of capped transcripts following TgCdk7 inhibition was observed. Parasites were harvested at time 0 and at 1 and 2 h after treatment with 10 μM of BS-181. RNA-IP with anti-cap (m3G-cap & m7G-cap) antibody followed by quantitative RT-PCR using TUB1 and ACT1-specific primers showed marked reduction in 5′-capping of TUB1 and ACT1 transcripts after 2 h of inhibitor treatment.