Interphase cytosol extracts prepared from Xenopus laevis eggs are active in RNA polymerase III (Pol III) transcription. Addition of recombinant B1 cyclin to these extracts activates mitotic protein kinases that repress transcription. Affinity-purified p34cdc2-cyclin B kinase (mitosis-promoting factor) is sufficient to effect this repression in a simplified Pol III transcription system. This mitotic repression involves the direct phosphorylation of a component of the Pol III transcription initiation factor TFIIIB, which consists of the TATA box-binding protein (TBP) and associated Pol III-specific factors. The transcriptional activity of the TFIIIB-TBP fraction can be modulated in vitro by phosphorylation with mitotic kinases and by dephosphorylation with immobilized alkaline phosphatase.

Abstract. A normal consequence of mitosis in eukaryotes is the repression of transcription. UsingXenopus egg extracts shifted to a mitotic state by the addition of purified cyclin, we have for the first time been able to reproduce a mitotic repression of transcription in vitro. Active RNA polymerase l/I transcription is observed in interphase extracts, but strongly repressed in extracts converted to mitosis. With the topoisomerase II inhibitor VM-26, we demonstrate that this mitotic repression of RNA polymerase III transcription does not require normal chromatin condensation. Similarly, in vitro mitotic repression of transcription does not require the presence of nucleosome structure or involve a general repressive chromatin-binding protein, as i~bition of chromatin formation with saturating amounts of non-specific DNA has no effect on repression. Instead, the mitotic repression of transcription appears to be due to phosphorylation of a component of the transcription machinery by a mitotic protein kinase, either cdc2 kinase and/or a kinase activated by it. Mitotic repression of RNA polymerase Ill transcription is observed both in complete mitotic cytosol and when a kinase-enriched mitotic fraction is added to a highly simplified 5S RNA transcription reaction. We present evidence that, upon depletion of cdc2 kinase, a secondary protein kinase activity remains and can mediate this in vitro mitotic repression of transcription.

Transcription factor IIIA (TFIIIA) from Xenopus oocytes binds both the internal control region of the 5S ribosomal RNA genes and the 5S RNA transcript itself. The nucleic acid binding domain of TFIIIA contains nine tandemly repeated zinc finger motifs. A series of precisely truncated forms of this protein have been constructed and assayed for 5S RNA and DNA binding. Different sets of zinc fingers were found to be responsible for high affinity interactions with RNA and with DNA. These results explain how a single protein can exhibit equal affinities for these two very different nucleic acids.

A series of polypeptides containing increasing numbers of zinc fingers of Xenopus trinscription factor MA has been generated and binding to the 5S RNA gene internal control region has been studied in order to elucidate the mode of interaction of the individual fingers with DNA. By using a combination of DNase I footprinting, methylation interference, and differential binding to mixtures of DNA fragments differing in length by single base pairs, the binding sites for individual fingers have been defined. These results have led to a model for the interaction of transcription factor MIlA with the internal control region in which fingers 1-3 bind in the major groove of the promoter C block, fingers 7-9 bind in the major groove of the A block, and finger 5 binds in the major groove of the intermediate element. Fingers 4 and 6 each bind across the minor groove, spanning these promoter elements.

Many aspects of transcriptional regulation in eukaryotic cells involve reversible phosphorylation events; these include phosphorylation of both general and gene-specific transcription factors (20, 21). Examples of both positive and negative effects of protein phosphorylation have been reported (for reviews, see references 20 and 21). One such example of transcriptional control mediated by phosphorylation is the global repression of nuclear transcription that occurs when cells enter mitosis (22,36). Over the years, numerous hypotheses have been put forward to explain mitotic repression of transcription (for reviews, see references 12, 16, and 32), including condensation of interphase chromatin into mitotic chromosomes, dissociation of transcription factors or RNA polymerase from the chromatin template, and inactivation of the basal transcription machinery by protein phosphorylation (13,40,50). For the genes transcribed by RNA polymerase III (pol III) (such as 5S rRNA and tRNA genes), previous studies have documented that the activity of the general class III transcription factor TFIIIB is greatly diminished in extracts from synchronized mitotic cells (49) or by the conversion of an interphase Xenopus egg extract to the mitotic state by the addition of recombinant cyclin B1 protein (13,16,50). In the latter experiments, the recombinant cyclin formed a complex with the p34 cdc2 kinase subunit present in the extract and, after a series of specific phosphorylation and dephosphorylation events, the active form of the cdc2/cyclin B kinase (maturation-mitosis promoting factor) was generated (45,46). Inhibition of transcription has been shown to be due to the enzymatic action of this kinase on a TFIIIB subunit (or a repressor protein that binds to and inactivates TFIIIB) (13, 16).Similar to class III gene transcription, transcription of mRNA-coding genes by RNA polymerase II (pol II) is also repressed at mitosis. We have shown that purified cdc2/cyclin B kinase is sufficient to inhibit transcription by pol II in a reconstituted transcription system (27). Recently, Segil et al. (40) reported that the general pol II transcription factor TFIID isolated from mitotic cells is multiply phosphorylated and inactive in supporting activator-dependent transcription. TFIID is composed of the TATA-binding protein (TBP) and TBPassociated factors (TAFs), and the TAFs have been shown to be involved in activator-dependent transcription (for reviews, see references 17 and 33). The activity of mitotic TFIID can be restored by dephosphorylation, showing that a protein phosphorylation event regulates TFIID during mitosis. Thus, for both activated pol II transcription (40) and pol III transcription (13, 49), a TBP-associated factor is inactivated at mitosis. In the work of Segil et al. (40), only TFIID was purified from mitotic cells; thus, it is not clear whether other targets of mitotic regulation exist in the pol II transcription machinery. Indeed, mitotic TFIID was found to be defective in only activator-dependent transcription, suggest...