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

Abstract

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-beta-D-ribofuranosylbenzimidazole (DRB), we have visualized the chromosomal distribution of RNA polymerase II (pol II) with hypophosphorylated (pol IIA) and hyperphosphorylated (pol II0) carboxyl-terminal repeat domain (CTD). DRB blocks labeling of the CTD with 32Pi within minutes of its addition, and nuclear pol II0 is gradually converted to IIA; this conversion parallels the reduction in transcription of protein-coding genes. DRB also alters the chromosomal distribution of II0: 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 II0 is coupled to the completion of nascent pol II gene transcripts.

Modulation of RNA Polymerase II Elongation Efficiency by C-terminal Heptapeptide Repeat Domain Kinase I

“…DRB and H8 inhibit CTD phosphorylation in vivo (29,30) and in vitro (2,31) and have been suggested to be inhibitors of transcription factor TFIIH (however see Ref. 18).…”

Control of RNA Polymerase II Elongation Potential by a Novel Carboxyl-terminal Domain Kinase

“…After the initial transition into productive elongation has been passed, it may be necessary to maintain the highly phosphorylated state of the polymerase. However, the results of Egyhazi et al (66) and Kephart et al (21) suggest that if CTD kinases are needed for maintenance of the IIO form during elongation then these kinases are not sensitive to DRB. Therefore, P-TEFb is not likely involved in maintenance.…”

“…Dubois et al (65) found that the addition of DRB or H-8 to HeLa cells caused a rapid decrease in the amount of RNA polymerase IIO. Egyhazi et al (66) recently showed that DRB inhibited the phosphorylation of RNA polymerase II to the IIO form in Chironomus tentans. Their results indicated that addition of DRB immediately stopped the incorporation of phosphate into the polymerase but that polymerases in productive elongation complexes maintained their hyperphosphorylated states until they completed their current round of transcription.…”

RNA Polymerase II Elongation Control

“…Other elongation factors come into the complex and provide it with the remarkable ability to elongate at a steady ~4 kb/minute through all obstacles for up to 2 million bp (96). The continued kinase activity of P-TEFb is not needed to maintain this rate (97). The identity and exact function of all of these factors is not known, but a number of candidate factors have been described.…”