PubMed Health⌕ Search

Biomedical subjects

Robert Schleif

Publications and source records attributed to Robert Schleif.

6 recordsLinked to original sources
PubMed2004

A portable allosteric mechanism.

The allosteric mechanism by which the gene expression regulatory protein AraC regulates its DNA-binding activity is shown to be portable by grafting it to beta-galactosidase, generating an arabinose-regulated beta-galactosidase. A portion of the alpha-peptide sequence that complements the activity of alpha-acceptor beta-galactosidase was inserted into a nonessential region of the regulatory peptidyl arm of AraC protein. Arabinose, which regulates the position of the arm in AraC protein now regulates the availability of the alpha-peptide to alpha-acceptor beta-galactosidase, thereby modulating its activity in response to arabinose.

Allosteric Regulation↗
PubMed2004

Biochemical and physiological properties of the DNA binding domain of AraC protein.

Intact AraC protein is poorly soluble and difficult to purify, whereas its dimerization domain is the opposite. Unexpectedly, the DNA binding domain of AraC proved also to be soluble in cells when overproduced and is easily purified to homogeneity. The DNA binding affinity of the DNA binding domain for its binding site could not be measured by electrophoretic mobility shift because of its rapid association and dissociation rates, but its affinity could be measured with a fluorescence assay and was found to have a dissociation constant of 1 x 10(-8)M in 100 mM KCl. The binding of monomers of the DNA binding domain to adjacent half-sites occurs without substantial positive or negative cooperativity. A simple analysis relates the DNA binding affinities of monomers of DNA binding domain and normal dimeric AraC protein.

AraC Transcription Factor↗
PubMed2004

Arm-domain interactions can provide high binding cooperativity.

Peptidyl arms extending from one protein domain to another protein domain mediate many important interactions in biology. A well-studied example of this type of protein-protein interaction occurs between the yeast homeodomain proteins, MAT alpha2 and MAT a1, which form a high-affinity heterodimer on DNA. The carboxyl-terminal arm extending from MAT alpha2 to MAT a1 has been proposed to produce an allosteric conformational change in the a1 protein that generates a very large increase in the DNA binding affinity of a1. Although early studies lent some support to this model, a more recent crystal structure determination of the free a1 protein argues against any allosteric change. This note presents a thermodynamic argument that accounts for the proteins' binding behavior, so that allosteric conformational changes are not required to explain the large affinity increase. The analysis presented here should be useful in analyzing binding behavior in other systems involving arm interactions.

Computational Biology↗
PubMed2003

Mutational analysis of residue roles in AraC function.

The previously isolated hemiplegic, induction-negative, repression-positive mutants, H80R and Y82C, were found to be defective in the binding of arabinose. Randomization of other residues close to arabinose in the three-dimensional structure of AraC or that make strong interactions with arabinose yielded induction-negative, repression-positive mutants. The induction and repression properties of mutants obtained by randomizing individual residues of the N-terminal arm of AraC allowed identification of the domain with which that residue very likely makes its predominant interactions. Residues 8-14 of the arm appear to make their predominant interaction with the DNA-binding domain. Although the side-chain of residue 15 interacts directly with arabinose bound to the N-terminal dimerization domain, the properties of mutant F15L indicate that this mutation increases the affinity of the arm for the DNA-binding domain.

AraC Transcription Factor↗
PubMed2003

AraC protein: a love-hate relationship.

In the bacterium Escherichia coli, the AraC protein positively and negatively regulates expression of the proteins required for the uptake and catabolism of the sugar L-arabinose. This essay describes how work from my laboratory on this system spanning more than thirty years has aided our understanding of positive regulation, revealed DNA looping (a mechanism that explains many action-at-a-distance phenomena) and, more recently, has uncovered the mechanism by which arabinose shifts AraC from a state where it prefers to bind to two well-separated DNA half-sites and form a DNA loop to a state where it binds to two adjacent half-sites and activates transcription. This work required learning how to assay, purify, and work with a protein possessing highly uncooperative biochemical properties. Present work is focussed on understanding arabinose-responsive mechanism in atomic detail and is also directed towards understanding protein structure and function well enough to be able to engineer the allosteric mechanism seen in AraC onto other proteins.

AraC Transcription Factor↗