Wolf V J, Dang T, Hartl P, Gottesfeld J M
Department of Molecular Biology, Scripps Research Institute, La Jolla, California 92037.
Mol Cell Biol. 1994 Jul;14(7):4704-11. doi: 10.1128/mcb.14.7.4704-4711.1994.
Transcription of 5S rRNA and tRNA genes by RNA polymerase III (pol 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 pol III transcription apparatus. We find that the maturation/mitosis-promoting factor kinase (MPF, p34cdc2-cyclin B) can directly mediate this repression in vitro. Affinity-purified MPF and immune complexes formed with antibodies to the protein subunits of MPF (p34cdc2 and cyclin B) retain both histone H1 kinase activity and the capacity to repress transcription in the reconstituted transcription system. Transcription complexes of oocyte-type 5S RNA genes and tRNA genes are quantitatively more sensitive to MPF repression than the corresponding transcription complexes of the somatic-type 5S RNA gene. The differential transcription of oocyte- and somatic-type genes observed during early Xenopus embryogenesis has been reproduced with the reconstituted transcription system and affinity-purified MPF. This differential transcription may be due to the instability of transcription complexes on the oocyte-type genes and the heightened sensitivity of soluble transcription factors to inactivation by mitotic phosphorylation. Our results suggest that MPF may play a role in vivo in the establishment of the embryonic pattern of pol III gene expression.
在未受精的非洲爪蟾卵的胞质提取物以及源自非洲爪蟾卵母细胞的重构系统中,RNA聚合酶III(pol III)对5S rRNA和tRNA基因的转录受到一种或多种有丝分裂蛋白激酶的抑制。这种抑制是由于pol III转录装置的一个组分发生了磷酸化。我们发现,成熟/有丝分裂促进因子激酶(MPF,p34cdc2 - 细胞周期蛋白B)在体外可直接介导这种抑制作用。亲和纯化的MPF以及与针对MPF蛋白亚基(p34cdc2和细胞周期蛋白B)的抗体形成的免疫复合物,在重构转录系统中既保留组蛋白H1激酶活性,又具备抑制转录的能力。卵母细胞型5S RNA基因和tRNA基因的转录复合物对MPF抑制的敏感性在数量上比体细胞型5S RNA基因的相应转录复合物更高。利用重构转录系统和亲和纯化的MPF,重现了非洲爪蟾胚胎早期发育过程中观察到的卵母细胞型和体细胞型基因的差异转录。这种差异转录可能是由于卵母细胞型基因上转录复合物的不稳定性以及可溶性转录因子对有丝分裂磷酸化失活的高度敏感性所致。我们的结果表明,MPF可能在体内对建立pol III基因表达的胚胎模式发挥作用。