Young L S, Rivier D H, Sprague K U
Institute of Molecular Biology, University of Oregon, Eugene 97403.
Mol Cell Biol. 1991 Mar;11(3):1382-92. doi: 10.1128/mcb.11.3.1382-1392.1991.
We have examined the interaction of transcription factors TFIIIC and TFIIID with a silkworm alanine tRNA gene. Previous functional analysis showed that the promoter for this gene is unusually large compared with the classical tRNA promoter elements (the A and B boxes) and includes sequences downstream from the transcription termination site. The goal of the experiments reported here was to determine which sequences within the full promoter make stable contacts with transcription factors. We show that when TFIIIC and TFIIID are combined, a complex is formed with the tRNA(Ala)C gene. Neither factor alone can form this complex. DNase I digestion of gene-factor complexes reveals that most of the tRNA(Ala)C promoter is in contact with factors. The protected region extends from -1 to at least +136 and includes both the A and B boxes and the previously identified downstream promoter sequences. Analysis of mutant promoters shows that sequence-specific contacts throughout the protected region are required for binding. The role of 3'-flanking sequences in transcription factor binding explains the contribution of these sequences to the tRNA(Ala)C promoter. We discuss the possibility that such sequences affect promoter strength in other tRNA genes.
我们研究了转录因子TFIIIC和TFIIID与家蚕丙氨酸tRNA基因的相互作用。先前的功能分析表明，与经典的tRNA启动子元件（A盒和B盒）相比，该基因的启动子异常大，并且包括转录终止位点下游的序列。本文报道的实验目的是确定完整启动子内哪些序列与转录因子形成稳定的接触。我们发现，当TFIIIC和TFIIID结合时，会与tRNA（Ala）C基因形成一个复合物。单独的任何一个因子都无法形成这种复合物。对基因-因子复合物进行DNase I消化显示，tRNA（Ala）C启动子的大部分区域都与因子接触。受保护区域从-1延伸至至少+136，包括A盒和B盒以及先前鉴定出的下游启动子序列。对突变启动子的分析表明，整个受保护区域的序列特异性接触对于结合是必需的。3'侧翼序列在转录因子结合中的作用解释了这些序列对tRNA（Ala）C启动子的贡献。我们讨论了此类序列影响其他tRNA基因启动子强度的可能性。
