Kober I, Teichmann M, Seifart K H
Institut für Molekularbiologie und Tumorforschung, Lahnstrasse 3, Marburg, D-35033, Germany.
J Mol Biol. 1998 Nov 20;284(1):7-20. doi: 10.1006/jmbi.1998.2165.
It has been shown that under specific conditions, transcription of protein coding genes can be efficiently initiated by RNA polymerase (pol) III in vitro. We examined the formation and composition of such pol III transcription complexes on the duck histone H5 and alphaA-globin promoters and found that the essential step for the formation of pol III transcription complexes on these pol II promoters was the stable binding of transcription factor (TF) IIIB-beta. For this process, the intact TFIIIB-beta complex, consisting of TBP and associated factors (TAFs) was needed and the prior association of pol III assembly factors was not necessary. We demonstrate for the first time that hTFIIIB-beta alone is able to bind to pol II promoter DNA. This resulted in a very stable complex which was resistant to high concentrations of heparin. Although immunodepletion revealed that TBP is essentially required for complex formation, other components of hTFIIIB-beta must also be involved, since TBP itself is unable to form heparin-resistant complexes and does not mediate pol III commitment per se. pol III is recruited to these pol II promoters in a strictly TFIIIC1 dependent way. After binding of TFIIIB-beta, the addition of TFIIIC1 and pol III were sufficient to yield productive pol III transcription complexes, which utilized the correct pol II initiation site. From these findings, we postulate that TFIIIC1 is involved in the recruitment of pol III and may thus form a bridge between TFIIIB-beta and the enzyme. This finding provides the first evidence for functional contacts between TFIIIC1 and pol III, which could be of general importance for the assembly of pol III transcription complexes.
研究表明，在特定条件下，蛋白质编码基因的转录在体外可由RNA聚合酶（pol）III有效启动。我们检测了鸭组蛋白H5和αA - 珠蛋白启动子上此类pol III转录复合物的形成及组成，发现这些pol II启动子上形成pol III转录复合物的关键步骤是转录因子（TF）IIIB - β的稳定结合。对于此过程，需要由TBP和相关因子（TAFs）组成的完整TFIIIB - β复合物，且pol III组装因子的预先结合并非必要。我们首次证明单独的hTFIIIB - β能够结合到pol II启动子DNA上。这形成了一种非常稳定的复合物，对高浓度肝素具有抗性。尽管免疫去除实验表明TBP对于复合物形成至关重要，但hTFIIIB - β的其他成分也必定参与其中，因为TBP自身无法形成抗肝素复合物，且本身不介导pol III的起始。pol III以严格依赖TFIIIC1的方式被招募到这些pol II启动子上。TFIIIB - β结合后，添加TFIIIC1和pol III足以产生有活性的pol III转录复合物，该复合物利用正确的pol II起始位点。基于这些发现，我们推测TFIIIC1参与了pol III的招募，因此可能在TFIIIB - β和该酶之间形成一座桥梁。这一发现首次为TFIIIC1与pol III之间的功能联系提供了证据，这可能对pol III转录复合物的组装具有普遍重要性。
