Kassavetis George A, Grove Anne, Geiduschek E Peter
Division of Biological Sciences and Center for Molecular Genetics, University of California, San Diego, 9500 Gilman Drive, La Jolla, CA 92093-0634, USA.
EMBO J. 2002 Oct 15;21(20):5508-15. doi: 10.1093/emboj/cdf533.
Certain deletion mutants of the Brf1 and Bdp1 subunits of transcription factor (TF) IIIB retain the ability to recruit RNA polymerase (pol) III to its promoters, but fail to support promoter opening: deletions within an internal Bdp1 segment interfere with initiation of DNA strand separation, and an N-terminal Brf1 deletion blocks propagation of promoter opening past the transcriptional start site. The ability of DNA strand breaks to restore pol III transcription activity to these defective TFIIIB assemblies has been analyzed using U6 snRNA gene constructs. Breaks in a 21 bp segment spanning the transcriptional start rescue transcription in DNA strand-specific and subunit/mutation-specific patterns. A cluster of Bdp1 internal deletions also reverses the inactivation of transcription with wild-type TFIIIB generated by certain transcribed (template) strand breaks near the transcriptional start site. A structure-based model and topological considerations interpret these observations, explain how Bdp1 and Brf1 help to enforce the general upstream--> downstream polarity of promoter opening and specify requirements for polarity reversal.
转录因子（TF）IIIB的Brf1和Bdp1亚基的某些缺失突变体保留了将RNA聚合酶（pol）III招募至其启动子的能力，但无法支持启动子开放：Bdp1内部片段的缺失会干扰DNA链分离的起始，而Brf1的N端缺失会阻止启动子开放越过转录起始位点后的传播。使用U6 snRNA基因构建体分析了DNA链断裂将pol III转录活性恢复至这些有缺陷的TFIIIB组装体的能力。跨越转录起始位点的21 bp片段中的断裂以DNA链特异性和亚基/突变特异性模式挽救转录。一组Bdp1内部缺失也能逆转由转录起始位点附近某些转录（模板）链断裂产生的野生型TFIIIB导致的转录失活。基于结构的模型和拓扑学考虑解释了这些观察结果，说明了Bdp1和Brf1如何有助于强化启动子开放的一般上游→下游极性，并明确了极性逆转的要求。
