Crystallization and preliminary crystallographic analysis of the transcriptional regulator RfaH from Escherichia coli and its complex with ops DNA.
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 P6(1)22 or P6(5)22, with unit-cell parameters a = b = 45.46, c = 599.93 A. 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 P1 (unit-cell parameters a = 36.79, b = 44.01, c = 62.37 A, alpha = 80.62, beta = 75.37, gamma = 75.41 degrees ). Complete diffraction data sets were collected for RfaH and its complex with DNA at 2.4 and 1.6 A 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.
Vassylyeva
Marina N
MN
Department of Biochemistry and Molecular Genetics, University of Alabama at Birmingham, School of Medicine, 402B Kaul Genetics Building, 720 20th Street South, Birmingham, AL 35294, USA.
Svetlov
Vladimir
V
Klyuyev
Sergiy
S
Devedjiev
Yancho D
YD
Artsimovitch
Irina
I
Vassylyev
Dmitry G
DG
D003001:Cloning, Molecular; D003460:Crystallization; D018360:Crystallography, X-Ray; D004247:DNA / Q000737:chemistry* / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D009876:Operon; D010445:Peptide Elongation Factors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D015534:Trans-Activators / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D014158:Transcription, Genetic
D029968:Escherichia coli Proteins; D010445:Peptide Elongation Factors; C106401:RfaH protein, E coli; D015534:Trans-Activators; D004247:DNA