Westmark C J, Ghose R, Huber P W
Department of Chemistry and Biochemistry, University of Notre Dame, 251 Nieuwland Science Hall, Notre Dame,IN 46556-5670, USA.
Nucleic Acids Res. 1998 Oct 15;26(20):4758-64. doi: 10.1093/nar/26.20.4758.
Ribosomes prepared from somatic tissue of Xenopus laevis inhibit transcription by RNA polymerase III. This observation parallels an earlier report that a high speed fraction from activated egg extract, which is enrichedin ribosomes, inhibits RNA polymerase III activityand destabilizes putative transcription complexes assembled on oocyte 5S rRNA genes. Transcription of somatic- and oocyte-type 5S rRNA genes and a tRNA gene are all repressed in the present experiments. We find that 5S rRNA genes incubated in S150 extract prepared from immature oocytes exhibit an extensive DNase I protection pattern that is nearly identical to that of the ternary complex of TFIIIA and TFIIIC bound to a somatic 5S rRNA gene. The complexes formed in this extract are stable at concentrations of ribosomes that completely repress transcription, indicating that formation of the TFIII(A+C) complex is not the target of inhibition. Ribosomes taken through a high salt treatment no longer repress transcription of class III genes, establishing that the inhibition is due to an associated factor and not the particle itself. The inhibitory activity released from ribosomes is inactivated by treatment with proteinase K, but not micrococcal nuclease. Preincubation of ribosomes with a general protein kinase inhibitor, 6-dimethylaminopurine, eliminates repression of transcription. Western blot analysis demonstrates that p34(cdc2), which is known to mediate repression of transcription by RNA polymerase III, is present in these preparations of ribosomes and can be released from the particles upon extraction with high salt. These results establish that a kinase activity, possibly p34(cdc2), is the actual agent responsible for the observed inhibition of transcription by ribosomes.
从非洲爪蟾的体细胞组织中制备的核糖体可抑制RNA聚合酶III的转录。这一观察结果与早期的一份报告相似，该报告称，来自活化卵提取物的一个富含核糖体的高速组分可抑制RNA聚合酶III的活性，并使组装在卵母细胞5S rRNA基因上的假定转录复合物不稳定。在本实验中，体细胞型和卵母细胞型5S rRNA基因以及一个tRNA基因的转录均受到抑制。我们发现，在从未成熟卵母细胞制备的S150提取物中孵育的5S rRNA基因呈现出一种广泛的DNase I保护模式，该模式与结合在体细胞5S rRNA基因上的TFIIIA和TFIIIC三元复合物的模式几乎相同。在这种提取物中形成的复合物在完全抑制转录的核糖体浓度下是稳定的，这表明TFIII(A+C)复合物的形成不是抑制的靶点。经过高盐处理的核糖体不再抑制III类基因的转录，这表明抑制作用是由于一种相关因子而非颗粒本身。从核糖体释放的抑制活性经蛋白酶K处理后失活，但经微球菌核酸酶处理则不失活。用一种通用的蛋白激酶抑制剂6-二甲基氨基嘌呤对核糖体进行预孵育可消除转录抑制。蛋白质印迹分析表明，已知可介导RNA聚合酶III转录抑制的p34(cdc2)存在于这些核糖体制剂中，并且在用高盐提取时可从颗粒中释放出来。这些结果表明，一种激酶活性，可能是p34(cdc2)，是导致观察到的核糖体抑制转录的实际因子。
