Schneider G J, Sayre M H, Geiduschek E P
Department of Biology, University of California, San Diego, La Jolla 92093-0634.
J Mol Biol. 1991 Oct 5;221(3):777-94. doi: 10.1016/0022-2836(91)80175-t.
Transcription factor 1 (TF1) is the Bacillus subtilis phage SPO1-encoded member of the family of DNA-binding proteins that includes Escherichia coli HU and integration host factor, IHF. A gel electrophoretic retardation method has been used to show that a TF1 dimer binding to one of its preferred sites in (5-hydroxymethyl)uracil (hmUra)-containing DNA sharply bends the latter. In fact, the DNA-bending properties of TF1 and E. coli IHF are indistinguishable. Substitutions at amino acid 61 in the DNA-binding "arm" of TF1 are known to affect DNA-binding affinity and site selectivity. Experiments described here show that these substitutions also affect DNA bending. The selectivity of TF1 binding is very greatly diminished and the affinity is reduced when hmUra is replaced in DNA by thymine (T). An extension of the gel retardation method that permits an analysis of DNA bending by non-specifically bound TF1 is proposed. Under the assumptions of this analysis, the reduced affinity of TF1 for T-containing DNA is shown to be associated with bending that is still sharp. The analysis of the TF1-DNA interaction has also been extended by hydroxyl radical (.OH) and methylation interference footprinting at two DNA sites. At each of these sites, and on each strand, TF1 strongly protects three segments of DNA from attack by OH. Patches of protected DNA are centered approximately ten base-pairs apart and fall on one side of the B-helix. Methylation in either the major or minor groove in the central ten base-pairs of the two TF1 binding sites quantitatively diminishes, but does not abolish, TF1 binding. We propose that multiple protein contacts allow DNA to wrap around the relatively small TF1 dimer, considerably deforming the DNA B-helix in the process.
转录因子1（TF1）是枯草芽孢杆菌噬菌体SPO1编码的DNA结合蛋白家族成员，该家族包括大肠杆菌HU和整合宿主因子IHF。凝胶电泳阻滞法已被用于证明TF1二聚体与其在含（5-羟甲基）尿嘧啶（hmUra）的DNA中的一个优选位点结合会使后者急剧弯曲。事实上，TF1和大肠杆菌IHF的DNA弯曲特性难以区分。已知TF1的DNA结合“臂”中第61位氨基酸的取代会影响DNA结合亲和力和位点选择性。本文所述的实验表明，这些取代也会影响DNA弯曲。当DNA中的hmUra被胸腺嘧啶（T）取代时，TF1结合的选择性大大降低，亲和力也降低。本文提出了一种凝胶阻滞法的扩展方法，该方法允许分析非特异性结合的TF1引起的DNA弯曲。在该分析的假设下，TF1对含T的DNA亲和力降低与仍然尖锐的弯曲有关。TF1与DNA相互作用的分析也通过在两个DNA位点的羟基自由基（·OH）和甲基化干扰足迹法得到了扩展。在这些位点中的每一个以及每条链上，TF1都强烈保护三段DNA免受OH的攻击。受保护的DNA片段中心大约相隔十个碱基对，位于B-螺旋的一侧。两个TF1结合位点中央十个碱基对的大沟或小沟中的甲基化会定量降低但不会消除TF1的结合。我们提出多个蛋白质接触允许DNA缠绕相对较小的TF1二聚体，在此过程中使DNA B-螺旋发生相当大的变形。
