Analysis of heat shock element recognition by saturation mutagenesis of the human HSP70.1 gene promoter.

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.