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Transcriptional stimulation by the DNA binding protein Hap46/BAG-1M involves hsp70/hsc70 molecular chaperones.

ABSTRACT: The hsp70/hsc70-associating protein Hap46 of human origin, also called BAG-1M (Bcl-2-associated athanogene 1), has been characterized previously as a DNA binding protein, which is able to stimulate transcription. By use of in vitro assays we now show that Hap46-mediated transcriptional activation can occur from linearized as well as from supercoiled circular DNA and does not require the presence of a transcription promoter. Accordingly, we observed no preferential binding of Hap46 to overlapping DNA fragments covering the sequence of the cytomegalovirus (CMV) early promoter, thus suggesting non-specific binding. The C-terminal deletion variant Hap46DeltaC47, which is unable to associate with hsp70/hsc70 molecular chaperones, produced greatly diminished effects on transcription, indicating a significant involvement of hsp70/hsc70 chaperones but not an absolute requirement. In contrast, deletion of the acidic hexarepeat region, as in variant Hap46Delta12-62, did not disturb transcriptional stimulation. While full-length Hap46 readily formed complexes with a series of structurally unrelated transcription factors, variant Hap46DeltaC47 proved incapable of doing so. Together these data suggest that transcriptional stimulation is a major biological activity of Hap46 and point to involvement of hsp70/hsc70 molecular chaperones in transcription in concert with Hap46, thus providing a link between hsp70/hsc70 molecular chaperones and components of the transcription machinery.

SUBMITTER: Niyaz Y

PROVIDER: S-EPMC153731 | biostudies-literature | 2003 Apr

REPOSITORIES: biostudies-literature

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Transcriptional stimulation by the DNA binding protein Hap46/BAG-1M involves hsp70/hsc70 molecular chaperones.

Niyaz Yilmaz Y Frenz Irina I Petersen Gabriele G Gehring Ulrich U

Nucleic acids research 20030401 8

The hsp70/hsc70-associating protein Hap46 of human origin, also called BAG-1M (Bcl-2-associated athanogene 1), has been characterized previously as a DNA binding protein, which is able to stimulate transcription. By use of in vitro assays we now show that Hap46-mediated transcriptional activation can occur from linearized as well as from supercoiled circular DNA and does not require the presence of a transcription promoter. Accordingly, we observed no preferential binding of Hap46 to overlapping ...[more]

PMID: 12682371

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Project description:To help cells cope with protein misfolding and aggregation, Hsp70 molecular chaperones selectively bind a variety of sequences ("selective promiscuity"). Statistical analyses from substrate-derived peptide arrays reveal that DnaK, the E. coli Hsp70, binds to sequences containing three to five branched hydrophobic residues, although otherwise the specific amino acids can vary considerably. Several high-resolution structures of the substrate -binding domain (SBD) of DnaK bound to peptides reveal a highly conserved configuration of the bound substrate and further suggest that the substrate-binding cleft consists of five largely independent sites for interaction with five consecutive substrate residues. Importantly, both substrate backbone orientations (N- to C- and C- to N-) allow essentially the same backbone hydrogen-bonding and side-chain interactions with the chaperone. In order to rationalize these observations, we performed atomistic molecular dynamics simulations to sample the interactions of all 20 amino acid side chains in each of the five sites of the chaperone in the context of the conserved substrate backbone configurations. The resulting interaction energetics provide the basis set for deriving a predictive model that we call Paladin (Physics-based model of DnaK-Substrate Binding). Trained using available peptide array data, Paladin can distinguish binders and nonbinders of DnaK with accuracy comparable to existing predictors and further predicts the detailed configuration of the bound sequence. Tested using existing DnaK-peptide structures, Paladin correctly predicted the binding register in 10 out of 13 substrate sequences that bind in the N- to C- orientation, and the binding orientation in 16 out of 22 sequences. The physical basis of the Paladin model provides insight into the origins of how Hsp70s bind substrates with a balance of selectivity and promiscuity. The approach described here can be extended to other Hsp70s where extensive peptide array data is not available.

Dataset Information

Transcriptional stimulation by the DNA binding protein Hap46/BAG-1M involves hsp70/hsc70 molecular chaperones.

Publications

Transcriptional stimulation by the DNA binding protein Hap46/BAG-1M involves hsp70/hsc70 molecular chaperones.

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