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.

Modular recognition of 5-base-pair DNA sequence motifs by human heat shock transcription factor.

We investigated the recognition of the conserved 5-bp repeated motif NGAAN, which occurs in heat shock gene promoters of Drosophila melanogaster and other eukaryotic organisms, by human heat shock transcription factor (HSF). Extended heat shock element mutants of the human HSP70 gene promoter, containing additional NGAAN blocks flanking the original element, showed significantly higher affinity than the wild-type promoter element for human HSF in vitro. Protein-DNA contact positions were identified by hydroxyl radical protection, diethyl pyrocarbonate interference, and DNase I footprinting. New contacts in the mutant HSE constructs corresponded to the locations of additional NGAAN motifs. The pattern of binding indicated the occurrence of multiple DNA binding modes for HSF with the various constructs and was consistent with an oligomeric, possibly trimeric, structure of the protein. In contrast to the improved binding, the extended heat shock element mutant constructs did not exhibit dramatically increased heat-inducible transcription in transient expression assays with HeLa cells.

Sequences in E1A proteins of human adenovirus 5 required for cell transformation, repression of a transcriptional enhancer, and induction of proliferating cell nuclear antigen.

A range of deletion and other mutants in the coding region of the E1A gene of Ad5 has been assayed for transformation of baby rat kidney (BRK) cells in cooperation with ras, repression of the SV40 enhancer, and induction of proliferating cell nuclear antigen (PCNA). Transformation efficiency was drastically reduced by deletion of residues 4-25, 36-60, or 111-138 in exon 1 of the 289 residue (289R) and 243R E1A proteins. Deletion of other residues in exon 1 had little effect. With mutants in the region unique to the 289R protein, and in exon 2, the only effect on transformation seemed to be an increased tendency of mutant transformants, compared to wt, to migrate to form secondary foci. Repression assays, performed with E1A plasmids producing only the 243R protein, showed that deletion of residues 4-25 or 36-60 inhibited repression completely. Deletion of residues 128-138 reduced repression, but deletions elsewhere in exon 1 had little effect. Deletion of residues 188-204 in exon 2 reduced repression slightly, and deletion of all of exon 2 reduced it to about one-half. It is concluded that for transformation, there are two functional domains in E1A proteins, both in exon 1, both involved in binding different cellular proteins, and both probably concerned with different transforming functions. One of these domains, involving residues 4-25 and 36-60, also functions in repression, but the role of the second in repression is much less critical. All of the deletion mutants in exon 1 induced PCNA synthesis in BRK cells. This result, together with previously published work, suggests that the active site for PCNA induction either involves residues 61-69 or 82-85 in exon 1, which have not been deleted, or it does not depend on any single limited region of the E1A proteins.

Use of deletion and point mutants spanning the coding region of the adenovirus 5 E1A gene to define a domain that is essential for transcriptional activation.

To help in identifying functional domains within Ad5 E1A proteins, we have constructed a series of mutants that create deletions throughout these products. We have also produced several mis-sense point mutations in the unique 13 S mRNA region. These mutated E1A regions have been tested in plasmid form for their ability to activate transcription of an E3-promoted CAT gene. From the results, a major domain for transactivation has been identified. This begins between residues 138 and 147, ends between residues 188 and 204, and encompasses the unique 13 S region. This domain is sensitive to mis-sense mutations. Transactivation was unaffected by small deletions in the N-terminal half of E1A proteins between residues 4 and 138, but was destroyed when this whole region was deleted. The C-terminal 71 residues may affect transactivation, but the results with the mutant in which this region was deleted were variable. The results obtained with these mutants are discussed in relation to the transactivation obtained by J. W. Lillie et al. [(1987). Cell 50, 1091-1100] with a synthetic peptide similar to the domain described here.