Phosphorylation of some chromosomal nonhistone proteins in active genes is blocked by the transcription inhibitor 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB).

The distribution of rapidly phosphorylated chromosomal proteins between chromosome I, chromosome II + III, chromosome IV, and nuclear sap including the matrix was investigated in salivary gland cells of Chironomus tentans. Chromosome IV, which carries most active nonribosomal genes in the cell, was found to be enriched in four rapidly phosphorylated nonhistone polypeptides (Mr = 25,000, 30,000, 33,000, and 42,000) in parallel with the transcriptional activity rather than with the DNA content of the chromosome. Also the histones H2A and H4 are rapidly phosphorylated but the phosphorylation is proportional to the DNA content of each chromosome sample. The 32P-labeled Mr = 42,000 polypeptide immunologically cross-reacted with an antibody elicited against the transcription stimulatory factor S-II isolated from Ehrlich ascites tumor cells (Sekimizu, K., D. Mizuno, and S. Natori, 1979, Exp. Cell Res., 124:63-72). In addition, indirect immunofluorescence studies on chromosome IV with antisera against the stimulatory factor II revealed a selective staining of the active gene loci. The incorporation of 32P into three chromosome IV nonhistone polypeptides, especially into the Mr = 42,000 polypeptide, was lowered by 70-85% shortly after administration of 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB), a likely inhibitor of heterogeneous nuclear RNA transcription at initiation level. The possibility of a causal relationship between inhibited phosphorylation of chromosomal proteins and blocked transcription of heterogeneous nuclear RNA genes by DRB is discussed.

Specific inhibition of hnRNA synthesis by 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole. Requirement of a free 3'-hydroxyl group, but not 2'- or 5'-hydroxyls.

Five structural analogues of 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB), all with modified sugar moieties, have been examined for their inhibitory activities on RNA transcription in salivary glands of Chironomus tentans. The well-known ability of the parent DRB at 65 microM concentration to selectively inhibit hnRNA/mRNA synthesis by approx. 90% was essentially abolished on methylation of the 3'-OH; but, at an overdose the analogue suppressed labeling of all RNA classes examined (hnRNA/mRNA, rRNA, 4-5 S RNA) by 70-80%. By contrast, the 2'-O-methyl derivative of DRB was almost as effective as DRB itself in blocking transcription of hnRNA/mRNA genes. Blocking of both the 2' and 3' hydroxyls (2',3'-O-isopropylidene-DRB) completely abolished inhibitory activity, irrespective of the concentration employed. The 5'-deoxy-5'-chloro derivative of DRB was only slightly less effective than the parent DRB. An unusual aspect of the activities of 2'-O-methyl-DRB and 5'-deoxy-5'-chloro-DRB was their ability to stimulate synthesis of 4-5 S RNA by 25-45%. Also investigated was the influence of the various analogues on the rate of formation of [3H]UTP from [3H]uridine used as an RNA precursor. The rate of such formation of [3H]UTP was suppressed 2-6-fold by treatment with 2'-O-methyl or 3'-O-methyl-DRB, but was unaffected by 5'-deoxy-5'-chloro-DRB or 5,6-dichloro-1-alpha-D-arabinofuranosylbenzimidazole. The overall data point to the importance of a free 3'-OH in the ribose moiety of DRB for selective inhibitory activity. The alpha-D-arabinofuranosyl analogue, although less selective in inhibition of RNA transcription, still exhibits about 50% of the activity of DRB.

The size distribution of poly(A) in newly synthesized and old Balbiani ring RNA.

The size distribution of newly synthesized and old poly(A) sequences on transcripts of the giant tissue specific puffs, Balbiani rings in salivary glands of Chironomus tentans has been determined. After labeling with [3H]adenosine, poly(A) containing Balbiani ring RNA(75S RNA) was selectively collected by means of a recently developed technique. This combines electrophoretic fractionation and affinity chromatography in one run by insertion of poly(U) immobilized in glass fiber filters in an agarose gel slab. The majority of short-term labeled poly(A) chains released from poly(A) containing 75S RNA molecules is distributed within a narrow size range migrating as one peak with a mean value of 103 +/- 2 nucleotides, which is probably the initial length of poly(A). The labeling pattern of ribonuclease resistant poly(A) stretches after chase with unlabeled adenosine displays a relatively broad and heterogeneous size spectrum from at least 20 to more than 100 nucleotides. The main peak of labeled adenylate core in newly formed poly(A) containing RNA of non-Balbiani ring origin is dispersed within a broader size range than that of Balbiani ring RNA and possesses an average value of 94 +/- 2 nucleotides. During chase conditions, the relative frequency of occurrence of poly(A) chains of 75S RNA in the size range of 100 nucleotides exhibits a significant decrease in parallel with a rather uniform gain in the size classes between 20--50 nucleotides. However, the results are inconsistent with the existence of an age-dependent shortening of poly(A) chains in the balbiani ring RNA. A significant portion of 75S RNA molecules remain associated with poly(A) segments which are essentially of original size even after 21 hr in the presence of unlabeled adenosine. This finding provides support for the possibility that the initiation of the poly(A) shortening in 75S RNA is a stochastic process.

The binding of the alpha subunit of protein kinase CK2 and RAP74 subunit of TFIIF to protein-coding genes in living cells is DRB sensitive.

In a previous report, we documented that a major portion of the nuclear protein kinase CK2alpha (CK2alpha) subunit does not form heterooligomeric structures with the beta subunit, but it binds tightly to nuclear structures in an epithelial Chironomus cell line. We report here that the CK2alpha, but not beta, subunit is co-localized with productively transcribing RNA polymerase II (pol II) on polytene chromosomes of Chironomus salivary gland cells. Likewise, the RAP74 subunit ofTFIIF, a potential substrate for CK2, is co-localized with pol II. The occupancies of chromosomes with the CK2alpha and RAP74 subunits are sensitive to DRB, an inhibitor of pol II-based transcription and the activity of CK2 and pol II carboxyl-terminal kinases. DRB alters the chromosomal distribution of the CK2alpha and RAP74 subunits: there is a time-dependent clearance from the chromosomes of CK2alpha and RAP74 subunits, which coincides in time the completion and release of preinitiated transcripts after addition of DRB. The results suggest that both the CK2alpha and RAP74 subunits travel with the elongating pol II molecules along the DNA template during the entire transcription cycle. No detectable re-association of CK2alpha and RAP74 with the promoters takes, however, place after the completion of the preinitiated transcripts in the presence of DRB. In contrast, the binding of hypophosporylated pol II and TFIIH to the active gene loci is not abolished by the DRB regimen. Our data are consistent with the possibility that in living Chironomus salivary gland cells, DRB interferes with the recruitment of TFIIF, but not of TFIIH, to the promoter by interference with the activity of the CK2alpha subunit enzyme and phosphorylation of RAP74 and thereby DRB blocks transcription initiation.

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-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.

Heat-shock-specific phosphorylation and transcriptional activity of RNA polymerase II.

The carboxyl-terminal domain (CTD) of the largest RNA polymerase II (pol II) subunit is a target for extensive phosphorylation in vivo. Using in vitro kinase assays it was found that several different protein kinases can phosphorylate the CTD including the transcription factor IIH-associated CDK-activating CDK7 kinase (R. Roy, J. P. Adamczewski, T. Seroz, W. Vermeulen, J. P. Tassan, L. Schaeffer, E. A. Nigg, J. H. Hoeijmakers, and J. M. Egly, 1994, Cell 79, 1093-1101). Here we report the colocalization of CDK7 and the phosphorylated form of CTD (phosphoCTD) to actively transcribing genes in intact salivary gland cells of Chironomus tentans. Following a heat-shock treatment, both CDK7 and pol II staining disappear from non-heat-shock genes concomitantly with the abolishment of transcriptional activity of these genes. In contrast, the actively transcribing heat-shock genes, manifested as chromosomal puff 5C on chromosome IV (IV-5C), stain intensely for phosphoCTD, but are devoid of CDK7. Furthermore, the staining of puff IV-5C with anti-PCTD antibodies was not detectably influenced by the TFIIH kinase and transcription inhibitor 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB). Following heat-shock treatment, the transcription of non-heat-shock genes was completely eliminated, while newly formed heat-shock gene transcripts emerged in a DRB-resistant manner. Thus, heat shock in these cells induces a rapid clearance of CDK7 from the non-heat-shock genes, indicating a lack of involvement of CDK7 in the induction and function of the heat-induced genes. The results taken together suggest the existence of heat-shock-specific CTD phosphorylation in living cells. This phosphorylation is resistant to DRB treatment, suggesting that not only phosphorylation but also transcription of heat-shock genes is DRB resistant and that CDK7 in heat shock cells is not associated with TFIIH.