Benjamin Lawrence R, Chung Hye-Jung, Sanford Suzanne, Kouzine Fedor, Liu Juhong, Levens David
Gene Regulation Section, Laboratory of Pathology, Center for Cancer Research, National Cancer Institute, Building 10, Room 2N106, Bethesda, MD 20892-1500, USA.
Proc Natl Acad Sci U S A. 2008 Nov 25;105(47):18296-301. doi: 10.1073/pnas.0803279105. Epub 2008 Nov 17.
The far upstream element (FUSE) binding protein (FBP), a single-stranded nucleic acid binding protein, is recruited to the c-myc promoter after melting of FUSE by transcriptionally generated dynamic supercoils. Via interactions with TFIIH and FBP-interacting repressor (FIR), FBP modulates c-myc transcription. Here, we investigate the contributions of FBP's 4 K Homology (KH) domains to sequence selectivity. EMSA and missing contact point analysis revealed that FBP contacts 4 separate patches spanning a large segment of FUSE. A SELEX procedure using paired KH-domains defined the preferred subsequences for each KH domain. Unexpectedly, there was also a strong selection for the noncontacted residues between these subsequences, showing that the contact points must be optimally presented in a backbone that minimizes secondary structure. Strategic mutation of contact points defined in this study disabled FUSE activity in vivo. Because the biological specificity of FBP is tuned at several layers: (i) accessibility of the site; (ii) supercoil-driven melting; (iii) presentation of unhindered bases for recognition; and (iv) modular interaction of KH-domains with cognate bases, the FBP-FIR system and sequence-specific, single-strand DNA binding proteins in general are likely to prove versatile tools for adjusting gene expression.
远上游元件（FUSE）结合蛋白（FBP）是一种单链核酸结合蛋白，在转录产生的动态超螺旋使FUSE解链后，它被招募到c-myc启动子上。通过与TFIIH和FBP相互作用阻遏物（FIR）相互作用，FBP调节c-myc转录。在此，我们研究了FBP的4个KH结构域对序列选择性的贡献。电泳迁移率变动分析（EMSA）和缺失接触点分析表明，FBP与跨越FUSE大片段的4个独立区域相互接触。使用成对KH结构域的SELEX程序确定了每个KH结构域的优选子序列。出乎意料的是，对这些子序列之间的非接触残基也有强烈的选择，这表明接触点必须在使二级结构最小化的主链中以最佳方式呈现。本研究中定义的接触点的策略性突变在体内使FUSE活性丧失。由于FBP的生物学特异性在几个层面上进行调节：（i）位点的可及性；（ii）超螺旋驱动的解链；（iii）用于识别的无阻碍碱基的呈现；以及（iv）KH结构域与同源碱基的模块化相互作用，一般而言，FBP-FIR系统和序列特异性单链DNA结合蛋白可能是调节基因表达的通用工具。
