Ghavidel A, Hockman D J, Schultz M C
Department of Biochemistry, University of Alberta, Edmonton, Canada.
Mol Cell Biochem. 1999 Jan;191(1-2):143-8.
We have investigated the molecular basis of the requirement for protein kinase CK2 in nuclear transcription in Saccharomyces cerevisiae. In vivo and in vitro analysis has demonstrated that CK2 is required for efficient transcription of the tRNA and 55 rRNA genes by RNA polymerase III. This suggests that a component of the pol III transcription machinery is regulated by CK2. We tested this possibility by a biochemical complementation approach in which components of the pol III transcription machinery from wild type cells were tested for their ability to rescue transcription in extract from a conditionally CK2-deficient mutant. We found that pol III transcription initiation factor IIIB (TFIIIB) fully restores transcription in CK2-deficient extract. Further in vitro studies revealed that TFIIIB must be phosphorylated to be active, that a single subunit of wild type TFIIIB, the TATA binding protein (TBP), is efficiently phosphorylated by CK2, and that recombinant TBP and a limiting amount of CK2 rescues transcription in CK2-deficient extract. We conclude that TBP is the physiological target of CK2 among the components of the pol III transcription machinery. The implications of this result are discussed in the context of previous data concerning the regulation of TFIIIB.
我们研究了酿酒酵母核转录过程中蛋白激酶CK2需求的分子基础。体内和体外分析表明，RNA聚合酶III高效转录tRNA和55 rRNA基因需要CK2。这表明pol III转录机制的一个组成部分受CK2调控。我们通过生化互补方法验证了这一可能性，即测试野生型细胞中pol III转录机制的组分拯救条件性CK2缺陷型突变体提取物中转录的能力。我们发现pol III转录起始因子IIIB（TFIIIB）能完全恢复CK2缺陷型提取物中的转录。进一步的体外研究表明，TFIIIB必须被磷酸化才能具有活性，野生型TFIIIB的单个亚基——TATA结合蛋白（TBP）能被CK2有效磷酸化，并且重组TBP和有限量的CK2能拯救CK2缺陷型提取物中的转录。我们得出结论，在pol III转录机制的组分中，TBP是CK2的生理靶点。结合先前关于TFIIIB调控的数据，讨论了该结果的意义。
