title: Low concentrations of free hydrophobic amino acids disrupt the Escherichia coli RNA polymerase core-sigma(70) protein-protein interaction.
abstract: Studies of the Escherichia coli RNA polymerase subunit sigma-70 employing limited proteolytic digestion and binding by monoclonal antibodies indicate that conserved region 3 is solvent accessible in the free protein and in the RNA polymerase holoenzyme. Conversely, when sigma-70 binds to core RNA polymerase, proteolytic cleavage of region 3 is dramatically reduced. The former set of results seems to indicate the physical presence of region 3 on or near the surface of the holoenzyme while the latter of these results suggest that region 3 is sequestered in a direct protein-protein contact within the RNA holoenzyme which alters its protease sensitivity. To further investigate these possibilities we inserted an internal histidine-tag within region 3 of sigma(70) (sigma(70)-R3-His6) between amino acids 508 and 509. Confirmation that the internal His-tag insertion does not disrupt normal sigma(70) function was verified by genetic complementation. His-tagged protein was immobilized on nickel-agarose and core RNAP was tethered via the sigma-core interaction. Our results are consistent with the localization of region 3 on or near the surface both of free sigma(70) and of RNA polymerase holoenzyme. Furthermore, we find that the sigma(70)-core interaction is resistant to high ionic conditions but is completely disrupted by the presence of the low-molecular-weight hydrophobic amino acids phenylalanine and leucine free in solution. These results demonstrate the general usefulness of this approach to the disruption of protein-protein interactions and its potential application for protein purification.
authors.affiliation: Department of Microbiology, Center of Microbial Pathogenesis, State University of New York at Buffalo School of Medicine, 14214, USA.
authors.forename: Amarnath
authors.initials: A
authors.lastname: Maitra
authors.forename: Jesus
authors.initials: J
authors.lastname: Moreno
authors.forename: V James
authors.initials: VJ
authors.lastname: Hernandez
mesh_terms: D003001:Cloning, Molecular; D012321:DNA-Directed RNA Polymerases / Q000737:chemistry* / Q000502:physiology; D004926:Escherichia coli; D006639:Histidine; D007930:Leucine / Q000737:chemistry*; D010649:Phenylalanine / Q000737:chemistry*; D011485:Protein Binding / Q000502:physiology; D012808:Sigma Factor / Q000737:chemistry* / Q000502:physiology
chemical_list: D012808:Sigma Factor; D010649:Phenylalanine; D006639:Histidine; C081775:RNA polymerase sigma 70; D012321:DNA-Directed RNA Polymerases; D007930:Leucine