The TFIIIC90 subunit of TFIIIC interacts with multiple components of the RNA polymerase III machinery and contains a histone-specific acetyltransferase activity.

Human transcription factor IIIC (hTFIIIC) is a multisubunit complex that directly recognizes promoter elements and recruits TFIIIB and RNA polymerase III. Here we describe the cDNA cloning and characterization of the 90-kDa subunit (hTFIIIC90) that is present within a DNA-binding subcomplex (TFIIIC2) of TFIIIC. hTFIIIC90 has no specific homology to any of the known yeast TFIIIC subunits. Immunodepletion and immunoprecipitation studies indicate that hTFIIIC90 is a bona fide subunit of TFIIIC2 and absolutely required for RNA polymerase III transcription. hTFIIIC90 shows interactions with the hTFIIIC220, hTFIIIC110, and hTFIIIC63 subunits of TFIIIC, the hTFIIIB90 subunit of TFIIIB, and the human RPC39 (hRPC39) and hRPC62 subunits of an initiation-specific subcomplex of RNA polymerase III. These interactions may facilitate both TFIIIB and RNA polymerase III recruitment to the preinitiation complex by TFIIIC. We show that hTFIIIC90 has an intrinsic histone acetyltransferase activity with a substrate specificity for histone H3.

Cloning and characterization of two evolutionarily conserved subunits (TFIIIC102 and TFIIIC63) of human TFIIIC and their involvement in functional interactions with TFIIIB and RNA polymerase III.

Human transcription factor IIIC (hTFIIIC) is a multisubunit complex that mediates transcription of class III genes through direct recognition of promoters (for tRNA and virus-associated RNA genes) or promoter-TFIIIA complexes (for the 5S RNA gene) and subsequent recruitment of TFIIIB and RNA polymerase III. We describe the cognate cDNA cloning and characterization of two subunits (hTFIIIC63 and hTFIIIC102) that are present within a DNA-binding subcomplex (TFIIIC2) of TFIIIC and are related in structure and function to two yeast TFIIIC subunits (yTFIIIC95 and yTFIIIC131) previously shown to interact, respectively, with the promoter (A box) and with a subunit of yeast TFIIIB. hTFIIIC63 and hTFIIIC102 show parallel in vitro interactions with the homologous human TFIIIB and RNA polymerase III components, as well as additional interactions that may facilitate both TFIIIB and RNA polymerase III recruitment. These include novel interactions of hTFIIIC63 with hTFIIIC102, with hTFIIIB90, and with hRPC62, in addition to the hTFIIIC102-hTFIIIB90 and hTFIIIB90-hRPC39 interactions that parallel the previously described interactions in yeast. As reported for yTFIIIC131, hTFIIIC102 contains acidic and basic regions, tetratricopeptide repeats (TPRs), and a helix-loop-helix domain, and mutagenesis studies have implicated the TPRs in interactions both with hTFIIIC63 and with hTFIIIB90. These observations further document conservation from yeast to human of the structure and function of the RNA polymerase III transcription machinery, but in addition, they provide new insights into the function of hTFIIIC and suggest direct involvement in recruitment of both TFIIIB and RNA polymerase III.

Enhanced transcriptional activation by E2 proteins from the oncogenic human papillomaviruses.

A systematic comparison of transcriptional activation by papillomavirus E2 proteins revealed that the E2 proteins from high-risk human papillomaviruses (human papillomavirus type 16 [HPV-16] and HPV-18) are much more active than are the E2 proteins from low-risk HPVs (HPV-6b and HPV-11). Despite the tropism of HPVs for particular epithelial cell types, this difference in transcriptional activation was observed in a number of different epithelial and nonepithelial cells. The enhanced activities of the E2 proteins from high-risk HPVs did not result from higher steady-state levels of protein in vivo, and in vitro DNA-binding assays revealed similar binding properties for these two classes of E2 proteins. These results demonstrate that the E2 proteins from high-risk HPVs have an intrinsically enhanced potential to activate transcription from promoters with E2-responsive elements. We found that there are also substantial differences between the activation properties of the bovine papillomavirus type 1 E2 protein and those of either of the two classes of HPV E2 proteins, especially with regard to requirements for particular configurations of E2 binding sites in the target promoter. Our results indicate that there are at least three distinct functional classes of E2 proteins and that these classes of E2 proteins may perform different roles during the respective viral life cycles.

Stockpiling of Cdc25 during a DNA replication checkpoint arrest in Schizosaccharomyces pombe.

The DNA replication checkpoint couples the onset of mitosis with the completion of S phase. It is clear that in the fission yeast Schizosaccharomyces pombe, operation of this checkpoint requires maintenance of the inhibitory tyrosyl phosphorylation of Cdc2. Cdc25 phosphatase induces mitosis by dephosphorylating tyrosine 15 of Cdc2. In this report, Cdc25 is shown to accumulate to a very high level in cells arrested in S. This shows that mechanisms which modulate the abundance of Cdc25 are unconnected to the DNA replication checkpoint. Using a Cdc2/cyclin B activation assay, we found that Cdc25 activity increased approximately 10-fold during transit through M phase. Cdc25 was activated by phosphorylations that were dependent on Cdc2 activity in vivo. Cdc25 activation was suppressed in cells arrested in G1 and S. However, Cdc25 was more highly modified and appeared to be somewhat more active in S than in G1. This finding might be connected to the fact that progression from G1 to S increases the likelihood that constitutive Cdc25 overproduction will cause inappropriate mitosis.

Cloning and characterization of a TFIIIC2 subunit (TFIIIC beta) whose presence correlates with activation of RNA polymerase III-mediated transcription by adenovirus E1A expression and serum factors.

TFIIIC2 is a general factor essential for transcription of 5S RNA, tRNA, and VA RNA genes by mammalian RNA polymerase III and consists of two forms designated TFIIIC2a and TFIIIC2b. TFIIIC2a and TFIIIC2b share common subunits of 220, 102, 90, and 63 kD but differ with respect to transcription activity and the presence of a presumptive 110-kD subunit in the active form (TFIIIC2a). Because both forms can bind the promoter directly, a selective role for the 110-kD subunit in the regulation of RNA polymerase III activity has been suggested. To investigate this possibility, we have cloned and expressed a cDNA encoding the 110-kD subunit (TFIIIC beta). Immunoprecipitation studies with anti-TFIIIC beta antibodies have confirmed that TFIIIC beta is a bona fide subunit present only in TFIIIC2a, that TFIIIC2a and the general factor TFIIIC1 are associated in unfractionated extracts, and that previously undetected polypeptides (potential TFIIIC1 subunits) can be isolated in association with TFIIIC2a. Previous studies have shown that increases in RNA polymerase III activity during infection of cells by adenovirus (with concomitant E1A expression) or during cell growth at high serum concentration results from an increased activity in the TFIIIC fraction. Studies with antibodies to TFIIIC beta have shown that this is strongly correlated with a selective increase in the cellular concentration of the TFIIIC beta 110-kD subunit and a concomitant rise in the ratio of the active-to-inactive forms of TFIIIC2.

Cloning and characterization of an evolutionarily divergent DNA-binding subunit of mammalian TFIIIC.

Transcription factor IIIC (TFIIIC) is required for the assembly of a preinitiation complex on 5S RNA, tRNA, and adenovirus VA RNA genes and contains two separable components, TFIIIC1 and TFIIIC2. TFIIIC2 binds to the 3' end of the internal control region of the VAI RNA gene and contains five polypeptides ranging in size from 63 to 220 kDa; the largest of these directly contacts DNA. Here we describe the cloning of cDNAs encoding all (rat) or part (human) of the 220-kDa subunit (TFIIIC alpha). Surprisingly, TFIIIC alpha has no homology to any of the yeast TFIIIC subunits already cloned, suggesting a significant degree of evolutionary divergence for RNA polymerase III factors. Antibodies raised against the N terminus of recombinant human TFIIIC alpha specifically inhibit binding of natural TFIIIC to DNA. Furthermore, immunodepletion assays indicate that TFIIIC alpha is absolutely required for RNA polymerase III transcription of 5S RNA, tRNA, and VAI RNA genes but not for the 7SK RNA and U6 small nuclear RNA genes. Transcription from the tRNA and VAI RNA genes in TFIIIC-depleted nuclear extracts can be restored by addition of purified TFIIIC. In contrast, restoration of 5S RNA gene transcription requires readdition of both TFIIIC and TFIIIA, indicating a promoter-independent interaction between these factors. Immunoprecipitation experiments demonstrate a tight association of all five polypeptides previously identified in the TFIIIC2 fraction, confirming the multisubunit structure of the human factor.

Purification and characterization of two forms of human transcription factor IIIC.

We showed previously that HeLa cell nuclear extracts contain two forms of transcription factor IIIC (TFIIIC) that formed chromatographically distinct TFIIIC-promoter complexes (Hoeffler, W. K., Kovelman, R., and Roeder, R. G. (1988) Cell 53, 907-920). One of these forms, the upper-band form, correlated with TFIIIC transcriptional activity, whereas the lower-band form bound to the VA1 promoter but supported little or no transcriptional activity. Using both transcription and DNA-binding assays, we have now purified both the upper-band form and the lower-band form of TFIIIC to near-homogeneity. The upper-band form is composed of five polypeptides with estimated sizes of 220, 110, 102, 90, and 63 kDa. The largest of these polypeptides can be cross-linked to the VA1 promoter. The lower-band form has a polypeptide structure similar to that of the upper-band form except for the absence or modification of the 110-kDa subunit. Direct assays show that the lower-band form is indeed transcriptionally inactive at all stages of purification, even when assayed with an unfractionated, heat-treated nuclear extract as a complementation system. This inactivity does not result from altered DNA-binding properties; instead, we suggest that the alteration of one of the subunits of TFIIIC renders it unable to interact productively with a downstream component of the transcription complex.

Sarkosyl defines three intermediate steps in transcription initiation by RNA polymerase III: application to stimulation of transcription by E1A.

We used Sarkosyl to analyze steps along the pathway of transcription initiation by RNA polymerase III. Sarkosyl (0.015%) inhibited transcription when present prior to incubation of RNA polymerase III, TFIIIB, and TFIIIC with the VAI gene, whereas it had no detectable effect on initiation or reinitiation of transcription when added subsequently. The formation of the corresponding 0.015% Sarkosyl-resistant complex required the presence of TFIIIC, TFIIIB, and RNA polymerase III but not nucleoside triphosphates. The addition of 0.05% Sarkosyl after this early step selectively inhibited a later step in the preinitiation pathway, allowing a single round of transcription after nucleoside triphosphate addition but blocking subsequent rounds of initiation. This step occurred prior to initiation because nucleoside triphosphates were not required for the formation of the corresponding 0.05% Sarkosyl-resistant complex. These observations provided a means to distinguish effects of regulatory factors on different steps in promoter activation and function. Using 0.05% Sarkosyl to limit reinitiation, we determined that the E1A-mediated stimulation of transcription by RNA polymerase III resulted from an increase in the number of active transcription complexes.

Activation of transcription factor IIIC by the adenovirus E1A protein.

The factor(s) responsible for the adenovirus E1A-stimulated transcription of RNA polymerase III genes was localized previously in a chromatographic fraction containing transcription factor IIIC (TFIIIC). In further studies, two distinct forms of TFIIIC, which were chromatographically separable, generated VA gene-protein complexes that were distinguished by gel shift assays. The form of TFIIIC that generated the more slowly migrating promoter complex had greater transcriptional activity in vitro, associated more rapidly with the promoter, and formed a more salt-resistant complex. Greater amounts of this more active form of TFIIIC resulted from either E1A expression during infection or growth of the cells in a higher concentration of serum, whereas template commitment assays indicated that overall TFIIIC concentrations remained unchanged during viral infection. The in vitro interconversion of the two forms of TFIIIC by phosphatase treatment suggests that transcriptional activation of RNA polymerase III genes can be mediated by phosphorylation of TFIIIC.