Cunniff N F, Morgan W D
Department of Biology, McGill University, Montreal, Quebec, Canada.
J Biol Chem. 1993 Apr 15;268(11):8317-24.
We analyzed the human HSP70.1 gene promoter heat shock element by saturation point mutagenesis and performed quantitative assays of in vitro heat shock factor binding and of in vivo transcription activity in HeLa cells with this extensive set of mutants. These results showed a significant correlation between measurements of heat shock factor binding and heat-inducible expression and provided a detailed thermodynamic description of the preferred recognition consensus sequence. In particular, this work demonstrated that outer positions 1 and 5 of the 5-base pair motif NGAAN, in addition to the most conserved triplet in the center, can have a strong influence on activity. Optimal activity occurred with the sequence AGAAC, and the levels of activity for all single base substitution variants were established. This analysis should be useful both for predicting the activity of potential heat shock element sequences near mammalian promoters and for interpreting structural features of protein-nucleic acid interactions in this system.
我们通过饱和点突变分析了人类HSP70.1基因启动子热休克元件，并利用这一整套突变体对HeLa细胞中的体外热休克因子结合及体内转录活性进行了定量测定。这些结果表明，热休克因子结合测量值与热诱导表达之间存在显著相关性，并提供了对优选识别共有序列的详细热力学描述。特别是，这项工作表明，5碱基基序NGAAN的外侧位置1和5，除了中心最保守的三联体外，也会对活性产生强烈影响。序列AGAAC出现最佳活性，并确定了所有单碱基替代变体的活性水平。该分析对于预测哺乳动物启动子附近潜在热休克元件序列的活性以及解释该系统中蛋白质-核酸相互作用的结构特征均应有所帮助。
