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.
Egyházi
E
E
Karolinska Institutet, Department of Cell and Molecular Biology, Stockholm, Sweden.
Ossoinak
A
A
Filhol-Cochet
O
O
Cochet
C
C
Pigon
A
A
D000818:Animals; D000918:Antibody Specificity; D047390:Casein Kinase II; D002683:Chironomidae / Q000235:genetics*; D008845:Microinjections; D011485:Protein Binding; D017346:Protein Serine-Threonine Kinases / Q000276:immunology / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D014157:Transcription Factors / Q000737:chemistry / Q000378:metabolism*; D019473:Transcription Factors, TFII
D011994:Recombinant Proteins; D014157:Transcription Factors; D019473:Transcription Factors, TFII; D047390:Casein Kinase II; D017346:Protein Serine-Threonine Kinases; C056604:transcription factor TFIIF