Crystallization and preliminary structure determination of Escherichia coli Mfd, the transcription-repair coupling factor

Transcription-repair coupling factors (TRCFs) are SF2 ATPases that couple transcription to DNA-damage repair by recognizing and removing RNA polymerase-elongation complexes stalled at DNA lesions and recruiting the nucleotide excision-repair machinery to the damaged sites. As a first step towards understanding the TRCF mechanism, the 130 kDa Escherichia coli TRCF ( the product of the mfd gene) has been overexpressed, purified and crystallized using an unusual precipitant, pentaerythritol ethoxylate. Initial phases were obtained using single-wavelength anomalous dispersion with a highly redundant 4 A resolution data set collected from selenomethionyl-substituted crystals and dramatically improved by density modification and phase extension to 3.2 angstrom resolution. Model building and refinement, which are in progress, will provide insight into transcription-coupled DNA-repair pathways, as this represents the first TRCF to be crystallized to date.