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Monoclonal antibodies that recognize specific carboxyl-terminal domain (CTD) phosphoepitopes were used to examine CTD phosphorylation in yeast cells lacking carboxyl-terminal domain kinase I (CTDK-I). We show that deletion of the kinase subunit CTK1 results in an increase in phosphorylation of serine in position 5 (Ser(5)) of the CTD repeat (Tyr(1)-Ser(2)-Pro(3)-Thr(4)-Ser(5)-Pro(6)-Ser(7)) during logarithmic growth. This result indicates that CTDK-I negatively regulates CTD Ser(5) phosphorylation. We also show that CTK1 deletion (ctk1Delta) eliminates the transient increase in CTD serine 2 (Ser(2)) phosphorylation observed during the diauxic shift. This result suggests that CTDK-I may play a direct role in phosphorylating CTD Ser(2) in response to nutrient depletion. Northern blot analysis was used to show that genes normally induced during the diauxic shift are not properly induced in a ctk1Delta strain. Glycogen synthase (GSY2) and cytosolic catalase (CTT1) mRNA levels increase about 10-fold in wild-type cells, but this increase is not observed in ctk1Delta cells suggesting that increased message levels may require Ser(2) phosphorylation. Heat shock also induces Ser(2) phosphorylation, but we show here that this change in CTD modification and an accompanying induction of heat shock gene expression is independent of CTDK-I. The observation that SSA3/SSA4 expression is increased in ctk1Delta cells grown at normal temperature suggests a possible role for CTDK-I in transcription repression. We discuss several possible positive and negative roles for CTDK-I in regulating CTD phosphorylation and gene expression.

Staphylococcal nuclease (nucleate 3'-oligonucleotidohydrolase; EC 3.1.4.7) cleaved DNA within disrupted adenovirus particles into a regular series of fragments with a repeat unit of 200 base pairs. Since this pattern did not eppear when DNA alone was digested, we postulate that the orderly arrangement of core polypeptides protects discrete regions of DNA from nuclease attack. The 23 X 10(6) dalton adenovirus DNA molecule can accommodate 180 units of roughly 200 base pairs. Based on the stoichiometry of core polypeptides, we calculate that each repeat unit contains six copies of polypeptide VII and a single copy of polypeptide V. This model is bases on proposals for the structure of eukaryotic chromatin. Very brief nuclease digestion generated 1800 base pair fragments (1/20 of the adenovirus chromosome). This result is discussed in terms of a higher order folding of viral DNA within the virus particle.

The largest subunit of RNA polymerase II contains a unique C-terminal domain (CTD) consisting of tandem repeats of the consensus heptapeptide sequence Tyr1-Ser2-Pro3-Thr4-Ser5-Pro6-Ser7. Two forms of the largest subunit can be separated by SDS-polyacrylamide gel electrophoresis. The faster migrating form termed IIA contains little or no phosphate on the CTD, whereas the slower migrating II0 form is multiply phosphorylated. CTD kinases with different phosphoryl acceptor specificities are able to convert IIA to II0 in vitro, and different phosphoisomers have been identified in vivo. In this paper we report the binding specificities of a set of monoclonal antibodies that recognize different phosphoepitopes on the CTD. Monoclonal antibodies like H5 recognize phosphoserine in position 2, whereas monoclonal antibodies like H14 recognize phosphoserine in position 5. The relative abundance of these phosphoepitopes changes when growing yeast enter stationary phase or are heat-shocked. These results indicate that phosphorylation of different CTD phosphoacceptor sites are independently regulated in response to environmental signals.

Transcriptional regulation by the androgen receptor (AR) requires its binding to hormone response element nucleotide sequences in DNA. A consensus glucocorticoid response element (GRE) can mediate transactivation by AR and other members of the AR/glucocorticoid (GR)/progesterone (PR)/mineralocorticoid (MR) receptor subfamily. We identified putative androgen response element (ARE) sequences by binding of a human AR DNA-binding domain fusion protein to DNA in a random sequence selection assay. A 17-base pair consensus nucleotide sequence, termed IDR17, containing three potential GRE-like core binding sites organized as both inverted and direct repeats, was determined from a pool of degenerate oligonucleotides. IDR17 was active in mediating androgen-dependent induction of reporter gene expression in transient transfection assays. Dissection of the IDR17 sequence revealed an 11-base pair sequence (DR-1), consisting of two potential core binding sites oriented as an overlapping direct repeat, as the most potent ARE. DR-1 demonstrated a strong preference for AR binding and transactivation when compared with GR. To our knowledge, this is the first observation that a direct repeat of GRE-like core motifs functions as a preferred hormone response element within the AR/GR/PR/MR subfamily of nuclear receptors.

RNA polymerase II is inhibited by the mushroom toxin alpha-amanitin. A mouse BALB/c 3T3 cell line was selected for resistance to alpha-amanitin and characterized in detail. This cell line, designated A21, was heterozygous, possessing both amanitin-sensitive and -resistant forms of RNA polymerase II; the mutant form was 500 times more resistant to alpha-amanitin than the sensitive form. By using the wild-type mouse RNA polymerase II largest subunit (RPII215) gene (J.A. Ahearn, M.S. Bartolomei, M. L. West, and J. L. Corden, submitted for publication) as the probe, RPII215 genes were isolated from an A21 genomic DNA library. The mutant allele was identified by its ability to transfer amanitin resistance in a transfection assay. Genomic reconstructions between mutant and wild-type alleles localized the mutation to a 450-base-pair fragment that included parts of exons 14 and 15. This fragment was sequenced and compared with the wild-type sequence; a single AT-to-GC transition was detected at nucleotide 6819, corresponding to an asparagine-to-aspartate substitution at amino acid 793 of the predicted protein sequence. Knowledge of the position of the A21 mutation should facilitate the study of the mechanism of alpha-amanitin resistance. Furthermore, the A21 gene will be useful for studying the phenotype of site-directed mutations in the RPII215 gene.

We have shown previously that UV radiation and other DNA-damaging agents induce the ubiquitination of a portion of the RNA polymerase II large subunit (Pol II LS). In the present study UV irradiation of repair-competent fibroblasts induced a transient reduction of the Pol II LS level; new protein synthesis restored Pol II LS to the base-line level within 16-24 h. In repair-deficient xeroderma pigmentosum cells, UV radiation-induced ubiquitination of Pol II LS was followed by a sustained reduction of Pol II LS level. In both normal and xeroderma pigmentosum cells, the ubiquitinated Pol II LS had a hyperphosphorylated COOH-terminal domain (CTD), which is characteristic of elongating Pol II. The portion of Pol II LS whose steady-state level diminished most quickly had a relatively hypophosphorylated CTD. The ubiquitinated residues did not map to the CTD. Importantly, UV-induced reduction of Pol II LS level in repair-competent or -deficient cells was inhibited by the proteasome inhibitors lactacystin or MG132. These data demonstrate that UV-induced ubiquitination of Pol II LS is followed by its degradation in the proteasome. These results suggest, contrary to a current model of transcription-coupled DNA repair, that elongating Pol II complexes which arrest at intragenic DNA lesions may be aborted rather than resuming elongation after repair takes place.

The carboxyl-terminal domain (CTD) of the mouse RNA polymerase II largest subunit consists of 52 repeats of a seven-amino-acid block with the consensus sequence Tyr-Ser-Pro-Thr-Ser-Pro-Ser. A genetic approach was used to determine whether the CTD plays an essential role in RNA polymerase function. Deletion, insertion, and substitution mutations were created in the repetitive region of an alpha-amanitin-resistant largest-subunit gene. The effects of these mutations on RNA polymerase II activity were assayed by measuring the ability of mutant genes to confer alpha-amanitin resistance after transfection of susceptible rodent cells. Mutations that resulted in CTDs containing between 36 and 78 repeats had no effect on the transfer of alpha-amanitin resistance, whereas mutations with 25 or fewer repeats were inactive in this assay. Mutations that contained 29, 31, or 32 repeats had an intermediate effect; the number of alpha-amanitin-resistant colonies was lower and the colonies obtained were smaller, indicating that the mutant RNA polymerase II was defective. In addition, not all of the heptameric repeats were functionally equivalent in that repeats that diverged in up to three amino acids from the consensus sequence could not substitute for the conserved heptamer repeats. We concluded that the CTD is essential for RNA polymerase II activity, since substantial mutations in this region result in loss of function.

The genomic locus (RPII215) encoding the largest subunit of mouse RNA polymerase II has been cloned by low stringency hybridization to a Drosophila RPII215 probe. The mouse gene consists of 28 exons which span 30 kilobases. Analysis of the nucleotide and predicted protein sequences indicates that the protein is comprised of two domains. There is a 1500 residue amino-terminal domain which contains seven regions strikingly similar to those in the beta' subunit of Escherichia coli RNA polymerase, and a carboxyl-terminal domain comprised of 52 repeats of a 7-amino-acid consensus sequence Tyr-Ser-Pro-Thr-Ser-Pro-Ser. Among the seven highly conserved regions are a strongly basic domain consistent with a DNA-binding site and a consensus sequence characteristic of a potential zinc-binding domain. The 5' upstream region contains three tandem sequences similar to binding sites for the transcription factor SP1. Two of the introns in this gene splice at donor GC dinucleotides as opposed to previously described invariant GT sites. The identification of regions which are highly conserved as compared with bacterial and yeast RNA polymerase and other regions which are unique to the mouse protein suggests which domains of RNA polymerase large subunits are involved in aspects of transcription common to both procaryotes and eucaryotes and which are characteristic of transcription in higher organisms.