Baumruker T, Sturm R, Herr W
Cold Spring Harbor Laboratory, New York 11724.
Genes Dev. 1988 Nov;2(11):1400-13. doi: 10.1101/gad.2.11.1400.
We have used the 100-kD HeLa cell octamer-binding protein OBP100 as a model to study flexible DNA sequence recognition by promoter-binding proteins. OBP100 binds to the conserved octamer motif ATGCAAAT found in numerous promoters and additionally to two degenerate octamer motifs (sites I and II) within the SV40 enhancer region. We show here that OBP100 binds the herpes simplex virus immediate early promoter TAATGARAT (R = purine) motif itself, extending the flexibility of OBP100 sequence recognition to sequences that bear very little resemblance (four matches over a 14-bp region). Nevertheless, a progression of OBP100-binding sites can be established that links the sequences of these two apparently unrelated binding sites by incremental steps. Mutational and chemical modification interference analyses of a degenerate octamer binding site (SV40 site II) show that specific sequences, which are not normally conserved but flank the degenerate octamer motif, can compensate for the degeneracy in the octamer core sequence. Thus, different regions of the binding site sequence (core or flanking) can diverge separately but not independently of one another. These results suggest that flexible DNA sequence recognition arises because there are few obligatory contact sites for OBP100 binding, but, rather, specific binding reflects the sum of many independent interactions.
我们以100-kD的HeLa细胞八聚体结合蛋白OBP100为模型，研究启动子结合蛋白对柔性DNA序列的识别。OBP100可与众多启动子中发现的保守八聚体基序ATGCAAAT结合，此外还能与SV40增强子区域内的两个简并八聚体基序（位点I和位点II）结合。我们在此表明，OBP100可结合单纯疱疹病毒立即早期启动子TAATGARAT（R = 嘌呤）基序本身，将OBP100序列识别的灵活性扩展到与之相似度极低的序列（在14个碱基对区域内有4个匹配）。然而，可以建立一个OBP100结合位点序列，通过逐步递增的方式将这两个明显不相关的结合位点的序列联系起来。对一个简并八聚体结合位点（SV40位点II）进行的突变和化学修饰干扰分析表明，通常不保守但位于简并八聚体基序侧翼的特定序列，可以补偿八聚体核心序列中的简并性。因此，结合位点序列的不同区域（核心或侧翼）可以分别发生变化，但并非彼此独立。这些结果表明，柔性DNA序列识别的产生是因为OBP100结合时几乎没有必需的接触位点，相反，特异性结合反映的是许多独立相互作用的总和。
