PubMed Health⌕ Search

Biomedical subjects

W Hendrickson

Publications and source records attributed to W Hendrickson.

24 records · Page 2Linked to original sources
PubMed1985

A dimer of AraC protein contacts three adjacent major groove regions of the araI DNA site.

Contact sites of AraC protein to the regulatory site araI of the Escherichia coli araBAD operon have been determined by the chemical-interference technique. DNA fragments were chemically modified an average of once per molecule, and fragments that no longer bound AraC were separated by gel electrophoresis from the DNA fragments still able to bind the protein. The contact sites were then determined by comparing the positions of modifications in the two DNA samples. Strong contacts were found with guanines in three consecutive major groove regions and the adjacent phosphates along one side of the DNA. The conserved bases of the AraC-binding DNA consensus sequence are also found in the same positions. The gel electrophoresis assay was used to determine the stoichiometry of binding, and AraC protein was found to bind the araI and araO1 regulatory sites as a dimer. Therefore, AraC appears to bind DNA differently from the other well-characterized regulatory proteins such as phage lambda repressor.

Alkylation↗
PubMed1984

Regulation of the Escherichia coli L-arabinose operon studied by gel electrophoresis DNA binding assay.

DNA binding properties of the proteins required for induction of the Escherichia coli L-arabinose operon were measured using a polyacrylamide gel electrophoresis assay. The mechanisms of induction and repression were studied by observing the multiple interactions of RNA polymerase, cyclic AMP receptor protein and araC protein with short DNA fragments containing either the araC or araBAD promoter regions. These studies show that binding of araC protein to the operator site, araO1, directly blocks RNA polymerase binding at the araC promoter, pC. We find that cyclic AMP receptor protein and araC protein do not bind co-operatively at their respective sites to linear DNA fragments containing the pBAD promoter. Nevertheless, both these positive effectors must be present on the DNA to stimulate binding of RNA polymerase. Additionally, binding of the proteins to the DNA is not sufficient; araC protein must also be in the inducing state, for RNA polymerase to bind. Equilibrium binding constraints and kinetics were determined for araC protein binding to the araI and the araO1 sites. In the presence of inducer, L-arabinose, araC protein binds with equal affinity to DNA fragments containing either of these sites. In the presence of anti-inducer, D-fucose, the affinity for both sites is reduced 40-fold. The apparent equilibrium binding constants for both states of the protein vary in parallel with the buffer salt concentration. This result suggests that the inducing and repressing forms of araC protein displace a similar number of cations upon binding DNA.

AraC Transcription Factor↗
PubMed1983

Spacing mutations between the Escherichia coli pBAD RNA polymerase binding site and the araC (I) induction site.

Mutations in the Escherichia coli promoter PBAD have been constructed which alter the spacing of the adjacent RNA polymerase and araC inducer protein binding sites. While deletion of a single base-pair or small insertions do not detectably affect araC protein binding to DNA and they do not alter the conserved sequence of the RNA polymerase binding site, stimulation of PBAD in vivo is greatly reduced. The experiments suggest that the distance or angle between the two proteins on the DNA is critical for promoter function.

AraC Transcription Factor↗
PubMed1980

Isolation of a replication origin complex from Escherichia coli.

A complex consisting of replicative origin DNA and several proteins was isolated from Escherichia coli. Cells of temperature-sensitive mutants were labeled at the origin and fractionated by sucrose gradient centrifugation. A complex highly purified in origin DNA sedimented as a unique band. This complex dissociated at high concentration, above 0.2 M KCl. Upon dialysis, the complex reformed, allowing further purification of its constituents. Three major protein bands were found, corresponding to proteins of the outer membrane. The complex did not sediment with membrane fractions, but adhered to the outer membrane in the presence of magnesium.

Bacterial Proteins↗