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 tenta ns, phosphorylation state-sensitive antibodies and the transcription a nd protein kinase inhibitor 5,6-dichloro-1-beta-D-ribofuranosyl-benzim idazole (DRB), we have visualized the chromosomal distribution of RNA polymerase II (pol II) with hypophosphorylated (pol IIA) and hyperphos phorylated (pol II0) carboxyl-terminal repeat domain (CTD). DRB blocks labeling of the CTD with P-32(i) within minutes of its addition, and nuclear pol II0 is gradually converted to IIA; this conversion paralle ls the reduction in transcription of protein-coding genes. DRB also al ters the chromosomal distribution of II0: there is a time-dependent cl earance from chromosomes of phosphoCTD (PCTD) after addition of DRB, w hich 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 chro mosomes with anti-CTD antibodies is not detectably influenced by the D RB treatment, indicating that hypophosphorylated pol IIA is unaffected by the transcription inhibitor. Microinjection of synthetic heptapept ide repeats, anti-CTD and anti-PCTD antibodies into salivary gland nuc lei hampered the transcription of BR2 genes, indicating the requiremen t for CTD and PCTD in transcription in living cells. The results demon strate that in vivo the protein kinase effector DRB shows parallel eff ects on an early step in gene transcription and the process of pol II hyperphosphorylation. Our observations are consistent with the proposa l that the initiation of productive RNA synthesis is CTD-phosphorylati on dependent and also with the idea that the gradual dephosphorylation of transcribing pol II0 is coupled to the completion of nascent pol I I gene transcripts.