Phosphorylation dependence of the initiation of productive transcription of Balbiani ring 2 genes in living cells.

Using polytene chromosomes of salivary gland cells of Chironomus tentans, phosphorylation state-sensitive antibodies and the transcription and protein kinase inhibitor 5,6-dichloro-1-β-d-ribofuranosyl-benzimidazole (DRB), we have visualized the chromosomal distribution of RNA polymerase II (pol II) with hypophosphorylated (pol IIA) and hyperphosphorylated (pol IIO) carboxyl-terminal repeat domain (CTD). DRB blocks labeling of the CTD with 32Pi within minutes of its addition, and nuclear pol IIO is gradually converted to IIA; this conversion parallels the reduction in transcription of protein-coding genes. DRB also alters the chromosomal distribution of IIO: there is a time-dependent clearance from chromosomes of phosphoCTD (PCTD) after addition of DRB, which coincides in time with the completion and release of preinitiated transcripts. Furthermore, the staining of smaller transcription units is abolished before that of larger ones. The staining pattern of chromosomes with anti-CTD antibodies is not detectably influenced by the DRB treatment, indicating that hypophosphorylated pol IIA is unaffected by the transcription inhibitor. Microinjection of synthetic heptapeptide repeats, anti-CTD and anti-PCTD antibodies into salivary gland nuclei hampered the transcription of BR2 genes, indicating the requirement for CTD and PCTD in transcription in living cells. The results demonstrate that in vivo the protein kinase effector DRB shows parallel effects on an early step in gene transcription and the process of pol II hyperphosphorylation. Our observations are consistent with the proposal that the initiation of productive RNA synthesis is CTD-phosphorylation dependent and also with the idea that the gradual dephosphorylation of transcribing pol IIO is coupled to the completion of nascent pol II gene transcripts.