Kassavetis George A, Steiner David F
Division of Biological Sciences and Center for Molecular Genetics, University of California-San Diego, 9500 Gilman Drive, La Jolla, CA 92093-0634, USA.
J Biol Chem. 2006 Mar 17;281(11):7445-51. doi: 10.1074/jbc.M512810200. Epub 2006 Jan 11.
The binding of the RNA polymerase III (pol III) transcription factor TFIIIC to the box A intragenic promoter element of tRNA genes specifies the placement of TFIIIB on upstream-lying DNA. In turn, TFIIIB recruits pol III to the promoter and specifies transcription initiating 17-19 base pairs upstream of box A. The resolution of the pol III transcription apparatus into recombinant TFIIIB, highly purified TFIIIC, and pol III is accompanied by a loss of precision in specifying where transcription initiation occurs due to heterogeneous placement of TFIIIB. In this paper we show that Nhp6a, an abundant high mobility group B (HMGB) family, non-sequence-specific DNA-binding protein in Saccharomyces cerevisiae restores transcriptional initiation fidelity to this highly purified in vitro system. Restoration of initiation fidelity requires the presence of Nhp6a prior to TFIIIB-DNA complex formation. Chemical nuclease footprinting of TFIIIC- and TFIIIB-TFIIIC-DNA complexes reveals that Nhp6a markedly alters the TFIIIC footprint over box A and reduces the size of the TFIIIB footprint on upstream DNA sequence. Analyses of unprocessed tRNAs from yeast lacking Nhp6a and its closely related paralogue Nhp6b demonstrate that Nhp6 is required for transcriptional initiation fidelity of some but not all tRNA genes, in vivo.
RNA聚合酶III（pol III）转录因子TFIIIC与tRNA基因的A框基因内启动子元件的结合决定了TFIIIB在上游DNA上的定位。反过来，TFIIIB将pol III招募到启动子上，并决定在A框上游17 - 19个碱基对处起始转录。由于TFIIIB定位的异质性，将pol III转录装置解析为重组TFIIIB、高度纯化的TFIIIC和pol III后，在确定转录起始位置时会出现精度损失。在本文中，我们表明Nhp6a（酿酒酵母中一种丰富的高迁移率族B（HMGB）家族的非序列特异性DNA结合蛋白）可恢复该高度纯化体外系统的转录起始保真度。起始保真度的恢复需要在TFIIIB - DNA复合物形成之前存在Nhp6a。对TFIIIC - 以及TFIIIB - TFIIIC - DNA复合物的化学核酸酶足迹分析表明，Nhp6a显著改变了A框上的TFIIIC足迹，并减小了上游DNA序列上的TFIIIB足迹大小。对缺乏Nhp6a及其密切相关的旁系同源物Nhp6b的酵母中未加工tRNA的分析表明，在体内，Nhp6是某些但不是所有tRNA基因转录起始保真度所必需的。
