Structure of MfdN and its influence on the stability and activity of the MFD protein
Abstract
Damage to actively transcribed genes is repaired at a faster rate than damage to non-transcribed genes due to the activity of proteins called transcription repair coupling factors. These proteins recognize RNA polymerases that have stalled on damaged DNA, remove them, and recruit DNA repair proteins to the site of the damage. In bacteria one protein performs all of those functions, Mfd. We are investigating the regulation of Mfd's activity and we hypothesize that the opening and closing of the interface between the protein's two principal domains, Mfd N (residues 1-450) and Mfd C (residues 473-1148), toggles Mfd between active and inactive states. We have solved the crystal structure of Mfd N by molecular replacement. Its structure superimposes well with that same region from the crystal structure of the full length Mfd protein (Deaconescu et al 2006), with an rmsd of 1.1 Å. The fold of Mfd N appears to be independent of Mfd C and the linker region (451-472), which is too disordered to be seen in the crystal structure. We have probed the stability of the N and C terminal regions of Mfd expressed as separate constructs, and the effect of disrupting the interface between them on the properties of Mfd. We believe that Mfd N exerts a stabilizing influence on structure of full length Mfd, and that it has an inhibitory effect on the protein's activity through its binding interactions with Mfd C.