A genetic and physical study of the interdomain linker ofE. ColiAraC protein-atrans-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.

Genome-Based Drug Target Identification in Human Pathogen Streptococcus gallolyticus

“…The interacting residues (green) are shown bonding (dotted lines) with the ligand. -López et al, 2015;Malaga et al, 2016). Table 11 presents the best results against AraC family transcriptional regulator (melR) where ZINC71781167 was predicted as top lead compound as shown in Figure 9.…”

Charged residues in the H-NS linker drive DNA binding and gene silencing in single cells

“…One demonstration of linker conformation effects on transcription is the AraC bacterial transcriptional regulator. In one conformation (suspected to be a random coil) AraC forms a repressive loop between distant molecules bound to DNA, whereas a change in linker conformation (proposed to be an increase in helicity) reorients the AraC dimer, and it binds to adjacent, activating sites on DNA (44). Recent studies have discovered that disordered proteins are abundant in proteomes across all domains of life and functionally significant.…”

Allosteric regulation within the highly interconnected structural scaffold of AraC/XylS homologs tolerates a wide range of amino acid changes

“…However, the structure of effector‐bound ToxT is monomeric 29 . Thus, information about inter‐domain and inter‐monomer contacts in full‐length AraC/XylS homodimers have largely been inferred from substitution experiments that diminish or enhance activity 31‐39 …”