
Crystallization and preliminary crystallographic analysis of the transcriptional regulator RfaH from Escherichia coli and its complex with ops DNA
Author(s) -
Vassylyeva Mari.,
Svetlov Vladimir,
Klyuyev Sergiy,
Devedjiev Yancho D.,
Artsimovitch Irina,
Vassylyev Dmitry G.
Publication year - 2006
Publication title -
acta crystallographica section f
Language(s) - English
Resource type - Journals
ISSN - 1744-3091
DOI - 10.1107/s174430910603658x
Subject(s) - crystallization , escherichia coli , dna , crystallography , biology , gene , nucleotide , transcription (linguistics) , chemistry , biophysics , microbiology and biotechnology , genetics , linguistics , philosophy , organic chemistry
The bacterial transcriptional factor and virulence regulator RfaH binds to rapidly moving transcription elongation complexes through specific interactions with the exposed segment of the non‐template DNA strand. To elucidate this unusual mechanism of recruitment, determination of the three‐dimensional structure of RfaH and its complex with DNA was initiated. To this end, the Escherichia coli rfaH gene was cloned and expressed. The purified protein was crystallized by the sitting‐drop vapor‐diffusion technique. The space group was P 6 1 22 or P 6 5 22, with unit‐cell parameters a = b = 45.46, c = 599.93 Å. A complex of RfaH and a nine‐nucleotide oligodeoxyribonucleotide was crystallized by the same technique, but under different crystallization conditions, yielding crystals that belonged to space group P 1 (unit‐cell parameters a = 36.79, b = 44.01, c = 62.37 Å, α = 80.62, β = 75.37, γ = 75.41°). Complete diffraction data sets were collected for RfaH and its complex with DNA at 2.4 and 1.6 Å resolution, respectively. Crystals of selenomethionine‐labeled proteins in both crystal forms were obtained by cross‐microseeding using the native microcrystals. The structure determination of RfaH and its complex with DNA is in progress.