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

Abstract

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 32 P-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 32 P 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-ß-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 .The nature ofevents involved in regulation ofeukaryotic gene expression at the transcriptional level is still to a large extent unknown . The recognition ofinitiation sites and the sequence ofevents involved in the step of transcription initiation play, in all likelihood, a crucial role in the exertion of gene control (for review, see reference 8). An important regulatory potential lies in the structure of chromatin . Unfolding of tightly packed chromatin fibers at selected gene loci may facilitate the finding of initiation sites by RNA polymerase molecules and transcription factors, and may guide the initiation reaction itself. Transcriptional regulatory potential may also lie in the concentration and chemical modification ofRNA polymerase and in specific transcription factors that may act independently on, or in cooperation with, RNA polymerase . Thus the identification ofmolecules and molecular assemblies with regulatory function and the elucidation of the significance of postsynthetic protein modifications as phosphorylation, acetylation, methylation, etc . in gene control require analysis of structural as well as catalytic constituents of the chromatin (for reviews, see references 42 and 46). On the basis of the involvement of posttranslational protein phosphorylation in many cellular regulatory mechanisms (for reviews, see references 20 and 37) and because of the observed modulation of specific chromosomal phosphoproteins in transcription...

Increased turnover of the messenger RNA encoding tyrosine aminotransferase can account for the desensitization and de-induction of tyrosine aminotransferase by 8-bromo-cyclic AMP treatment and removal.

“…The only previous direct measurement of the half-life of mRNATAT was 120 ± 18 min, with no apparent effect of a 3 h 8-Br-cAMP treatment (Spielholz et al, 1988). However, this study used the transcriptional inhibitor 5,6-dichlororibofuranosylbenzimidazole which has been shown to inhibit a protein kinase activity (Egyhazi et al, 1984;Zandomeni et al, 1986), alter the structure of mRNA protein particles (Dreyfuss et al, 1984), and may therefore affect RNA turnover. In order to measure mRNATAT turnover directly, without the use of inhibitors, we performed a [3H]uridine pulse-chase experiment.…”

Microinjection of anti-topoisomerase I immunoglobulin G into nuclei of Chironomus tentans salivary gland cells leads to blockage of transcription elongation.

“…Chromosome IV was prepared from isolated nuclei and used in the indirect immunofluorescence staining assay essentially as previously described (11,25,26). The chromosomes were fixed for 0 to 20 s. After staining, the chromosomes were mounted in the presence of p-phenylenediamine to retard fading during microscopy (20).…”

Posttranslational phosphorylation of specific chromosomal proteins and transcription of hnRNA genes in isolated nuclei: Retention of in vivo sensitivity to 5,6‐dichloro‐1‐β‐D‐ribofuranosylbenzimidazole (DRB)

“…We further found that transcriptionally active gene loci, especially the tissue-specific Balbiani rings, were enriched in rapidly phosphorylated 42-, 33-, 30-, and 25-kDa polypeptides [6]. Thus the phosphorylation of these proteins was correlated with the transcriptional activity of hnRNA genes.…”

“…Obviously, there are a variety of levels, including chromatin structure, recognition of promoter sites, initiation of transcription, etc, that may serve as targets for modification by addition of phosphate groups to specific nuclear proteins. The most suggestive support in favour of a causal relationship between phosphorylation and gene control comes from in vitro experiments demonstrating a stimulation of RNA polymerase I [3,4] as well as of polymerase I1 [4,5] activities by purified protein kinase NII, but correlative findings have also been obtained under in vivo assay conditions [6]. However, the establishment and characterization of the link between phosphorylation and gene regulation still remain to be done.…”

“…Thus the phosphorylation of these proteins was correlated with the transcriptional activity of hnRNA genes. Consequently, when hnRNA genes were inactivated at the level of chain initiation by the nucleoside analogue 5,6-dichloro-1-/3-D-ribofuranosylbenzimidazole (DRB), the transcription block coincided in time with the inhibition of the phosphorylation of 42-, 33-, and 30-kDa polypeptides [6]. Of special interest is the finding that the 42-kDa polypeptide is capable of crossreacting immunologically with antibodies raised against a transcription stimulatory factor derived from Ehrlich ascites tumor cells [6].…”