II) 1 (1, 2) and RNAP III (3), with RNAP II being the most sensitive. As a consequence, the incorporation of new ribonucleotides into the nascent RNA chains is blocked (4). Actinomycin D is generally thought to intercalate into DNA thereby preventing the progression of RNA polymerases, with RNAP I being the most sensitive (5, 6). In previous work, we have shown that the average phosphorylation of RNAP II C-terminal domain (CTD) increases in cells exposed to actinomycin D (7, 8). The activity of RNAP II is regulated by multisite phosphorylation on the CTD (9). The underphosphorylated CTD mediates multiple protein-protein interactions involved in the assembly of a preinitiation complex. The subsequent phosphorylation of the CTD occurs along the initiation of transcription and contributes to disrupt some of the interactions that lead to the assembly of the preinitiation complex on promoters. Phosphorylation of RNAP II at this step is required to elongate transcription and mediates the recruitment of various enzymatic complexes involved in processing of the primary transcript (10 -12). In contrast, phosphorylation of the CTD prior to the formation of the preinitiation complex represses the expression of specific genes (13). Hence, the increase in average phosphorylation of the CTD promoted by actinomycin D raises the possibility that different genes may have different susceptibilities to this drug.Several cyclin-dependent kinases (CDK) have been shown to phosphorylate the CTD and regulate transcription. CDK7, and its partner, cyclin H, are subunits of the general transcription factor, TFIIH, a component of the preinitiation complex (14, 15); CDK8 and its partner cyclin C belong to the RNAP II holoenzyme (16, 17); CDK9/PITALRE, and its partners, cyclins T1 and T2, are subunits of the transcription elongation factor P-TEFb (18). 5,6-Dichloro-1--D-ribofuranosylbenzimidazole (DRB) is another widely used transcriptional inhibitor (19) that inhibits CDK7 (20) and CDK9/PITALRE (21). The average CTD phosphorylation is decreased in cells exposed to DRB (7), suggesting that these kinases might contribute to global CTD phosphorylation in vivo.
The largest subunit of RNA polymerase II has an intriguing feature in its carboxyl-terminal domain (CTD) that consists of multiple repeats of an evolutionary conserved motif of seven amino acids. CTD phosphorylation plays a pivotal role in controlling mRNA synthesis and maturation. In exponentially growing cells, the phosphate turnover on the CTD is fast; it is blocked by common inhibitors of transcription, such as 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole and actinomycin D. Transcription-independent changes in CTD phosphorylation are observed at critical developmental stages, such as meiosis and early development.
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