title: A genetic and physical study of the interdomain linker of E. Coli AraC protein--a trans-subunit communication pathway.
abstract: Genetic experiments with full length AraC and biophysical experiments with its dimerization domain plus linker suggest that arabinose binding to the dimerization domain changes the properties of the inter-domain linker which connects the dimerization domain to the DNA binding domain via interactions that do not depend on the DNA binding domain. Normal AraC function was found to tolerate considerable linker sequence alteration excepting proline substitutions. The proline substitutions partially activate transcription even in the absence of arabinose and hint that a structural shift between helix and coil may be involved. To permit fluorescence anisotropy measurements that could detect arabinose-dependent dynamic differences in the linkers, IAEDANS was conjugated to a cysteine residue substituted at the end of the linker of dimerization domain. Arabinose, but not other sugars, decreased the steady-state anisotropy, indicating either an increase in mobility and/or an increase in the fluorescence lifetime of the IAEDANS. Time-resolved fluorescence measurements showed that the arabinose-induced anisotropy decrease did not result from an increase in the excited-state lifetime. Hence arabinose-induced decreases in anisotropy appear to result from increased tumbling of the fluorophore. Arabinose did not decrease the anisotropy in mutants incapable of binding arabinose nor did it alter the anisotropy when IAEDANS was conjugated elsewhere in the dimerization domain. Experiments with heterodimers of the dimerization domain showed that the binding of arabinose to one subunit of the dimer decreases the fluorescence anisotropy of only a fluorophore on the linker of the other subunit.
authors.affiliation: Biology Department, UPCH, Lima, San Martín De Porres, Peru.
authors.forename: Fabiana
authors.initials: F
authors.lastname: Malaga
authors.affiliation: Department of Biology, Johns Hopkins University, Baltimore, Maryland, 21218.
authors.forename: Ory
authors.initials: O
authors.lastname: Mayberry
authors.affiliation: Tufts University Medical School, Boston, Massachusetts.
authors.forename: David J
authors.initials: DJ
authors.lastname: Park
authors.affiliation: Department of Biology, Johns Hopkins University, Baltimore, Maryland, 21218.
authors.forename: Michael E
authors.initials: ME
authors.lastname: Rodgers
authors.affiliation: Department of Biology, Johns Hopkins University, Baltimore, Maryland, 21218.
authors.forename: Dmitri
authors.initials: D
authors.lastname: Toptygin
authors.affiliation: Department of Biology, Johns Hopkins University, Baltimore, Maryland, 21218.
authors.forename: Robert F
authors.initials: RF
authors.lastname: Schleif
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D051777:AraC Transcription Factor / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D001089:Arabinose / Q000737:chemistry* / Q000378:metabolism; D003545:Cysteine / Q000737:chemistry* / Q000378:metabolism; D004926:Escherichia coli / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D005454:Fluorescence Polarization; D015870:Gene Expression; D009154:Mutation; D009282:Naphthalenesulfonates / Q000737:chemistry; D011392:Proline / Q000737:chemistry* / Q000378:metabolism; D011485:Protein Binding; D000072417:Protein Domains; D017510:Protein Folding; D055503:Protein Multimerization; D021122:Protein Subunits / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D013816:Thermodynamics
chemical_list: D051777:AraC Transcription Factor; C497371:AraC protein, E coli; D029968:Escherichia coli Proteins; D009282:Naphthalenesulfonates; D021122:Protein Subunits; D011994:Recombinant Proteins; C006421:1,5-I-AEDANS; D011392:Proline; D001089:Arabinose; D003545:Cysteine
keywords: AraC protein; domain; fluorescence anisotropy; interdomain linker