Dieci G, Percudani R, Giuliodori S, Bottarelli L, Ottonello S
Institute of Biochemical Sciences, University of Parma, Parma, I-43100, Italy.
J Mol Biol. 2000 Jun 9;299(3):601-13. doi: 10.1006/jmbi.2000.3783.
The most peculiar transcriptional property of eukaryotic tRNA genes, as well as of other genes served by RNA polymerase III, is their complete dependence on the intragenic interaction platform provided by transcription factor IIIC (TFIIIC) for the productive assembly of the TBP-containing initiation factor TFIIIB. The sole exception, in yeast, is the U6 RNA gene, which is able to exploit a TATAAATA element, 30 bp upstream of the transcription start site, for the TFIIIC-independent assembly of TFIIIB. To find out whether this extragenic core promoter organization and autonomous TFIIIB assembly capacity are unique features of the U6 gene or also apply to other genes transcribed by RNA polymerase III, we scanned the 5'-flanking regions (up to position -100) of the entire tRNA gene set of Saccharomyces cerevisiae searching for U6-like TATA motifs. Four tRNA genes harboring such a sequence motif around position -30 were identified and found to be transcribed in vitro by a minimal system only composed of TFIIIB and RNA polymerase III. In this system, start site selection is not at all affected by the absence of TFIIIC, which, when added, significantly stimulates transcription by determining an increase in the number, rather than in the efficiency of utilization, of productive initiation complexes. A specific TBP-TATA element interaction is absolutely required for TFIIIC-independent transcription, but the nearby sequence context also contributes to the efficiency of autonomous TFIIIB assembly. The existence of a TFIIIB assembly pathway leading to the faithful transcription of natural eukaryotic tRNA genes in the absence of TFIIIC provides novel insights into the functional flexibility of the eukaryotic tRNA gene transcription machinery and on its evolution from an ancestral RNA polymerase III system relying on upstream, TATA- centered control elements.
真核生物tRNA基因以及由RNA聚合酶III负责转录的其他基因，其最独特的转录特性是它们完全依赖转录因子IIIC（TFIIIC）提供的基因内相互作用平台，来实现含TBP的起始因子TFIIIB的有效组装。在酵母中，唯一的例外是U6 RNA基因，它能够利用转录起始位点上游30 bp处的TATAAATA元件，进行不依赖TFIIIC的TFIIIB组装。为了弄清楚这种基因外核心启动子组织和自主TFIIIB组装能力是U6基因的独特特征，还是也适用于由RNA聚合酶III转录的其他基因，我们扫描了酿酒酵母整个tRNA基因集的5'侧翼区域（至-100位），寻找类似U6的TATA基序。我们鉴定出四个在-30位左右含有这种序列基序的tRNA基因，并发现它们在仅由TFIIIB和RNA聚合酶III组成的最小系统中能够进行体外转录。在这个系统中，起始位点的选择完全不受TFIIIC缺失的影响，而当加入TFIIIC时，它会通过增加有效起始复合物的数量而非利用效率来显著刺激转录。不依赖TFIIIC的转录绝对需要特定的TBP - TATA元件相互作用，但附近的序列背景也有助于自主TFIIIB组装的效率。在没有TFIIIC的情况下，存在一条导致天然真核生物tRNA基因忠实转录的TFIIIB组装途径，这为真核生物tRNA基因转录机制的功能灵活性及其从依赖上游以TATA为中心的控制元件的祖先RNA聚合酶III系统的进化提供了新的见解。
