Role of maturation-promoting factor (p34cdc2-cyclin B) in differential expression of the Xenopus oocyte and somatic-type 5S RNA genes.

Abstract

Transcription of 5S rRNA and tRNA genes by RNA polymerase III (pot III) in cytosolic extracts of unfertilized Xenopus eggs and in a reconstituted system derived from Xenopus oocytes is repressed by the action of one or more mitotic protein kinases. Repression is due to the phosphorylation of a component of the pot III transcription apparatus. We find that the maturation/mitosis-promoting factor kinase (MPF, The developmental regulation of transcription of the 5S RNA genes of the amphibian Xenopus laevis has been a subject of intensive investigation for almost 20 years (reviewed in reference 53). The Xenopus genome contains several multigene families that encode the 120-base-long 5S rRNA; the major oocyte-type gene family contains 20,000 copies per haploid genome, whereas the somatic-type gene family contains only 400 copies per genome equivalent (34). 5S genes of both families require similar sets of transcription factors for synthesis of 5S RNA by RNA polymerase III (pol III) in vitro (for reviews, see references 14, 32, and 50). These factors include the 5S gene-specific zinc finger protein TFIIIA and the general pol III factors TFIIIB and TFIIIC. TFIIIA binds to the internal control region of the 5S genes and directs the assembly of the other components of the transcription complex. TFIIIC is a large multisubunit protein which binds directly to the B-block promoter element of tRNA-like pol III genes. For the 5S genes (which lack a B-block sequence), TFIIIC binds to the TFIIIA-5S gene binary complex primarily through proteinprotein interactions (18). TFIIIB has recently been shown to consist of the TATA-box binding protein and pol III-specific TATA-binding protein-associated factors (21,26,45,47

Repression of TFIIH Transcriptional Activity and TFIIH-Associated cdk7 Kinase Activity at Mitosis

“…Xenopus mitotic cell extracts were generated by the addition of a recombinant sea urchin B1 cyclin-glutathione S-transferase (GST) fusion protein (46) to an interphase extract prepared from unfertilized eggs (16). Mitotic conversion was monitored by induction of histone H1 kinase activity (16,50). The activated cdc2/cyclin B kinase was recovered from the extract with p13-agarose beads or with glutathione-Sepharose beads as described previously (16,50).…”

“…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).…”

“…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).…”

Restricted Specificity of Xenopus TFIIIA for Transcription of Somatic 5S rRNA Genes

“…When Cdc2-cyclin B (MPF) was added to a reconstituted transcription system, the S/O ratio increased from 7 to 50 (77), which likely explains the apparent connection between Cdc2 activity and differential expression of the two 5S rRNA gene families. Our results suggest that the switch in 5S rRNA gene expression detected at the MBT results from phosphorylation events that occur during oocyte maturation.…”

A Two-Hit Mechanism for Pre-Mitotic Arrest of Cancer Cell Proliferation by a Polyamide-Alkylator Conjugate

“…Cdc2 kinase assays were carried out as previously described 32 with some modifications. Ten micrograms of either anticdc2 antibody (Santa Cruz Biotechnology, CA) or normal rabbit serum (Santa Cruz Biotechnology, CA) were coupled for 4 h at 4˚C on a rocker to protein A-sepharose 6 MB (Pharmacia), that was previously washed three times with 400 µL buffer A (12 mM Hepes, pH 7.5, 60 mM KCl, 6 mM MgCl 2 , and 8% glycerol).…”