Crystallization and preliminary crystallographic analysis of the transcriptional regulator RfaH from Escherichia coli and its complex with ops DNA
- Department of Biochemistry and Molecular Genetics, University of Alabama at Birmingham, Schools of Medicine and Dentistry, 402B Kaul Genetics Building, 720 20th Street South, Birmingham, AL 35294 (United States)
- Department of Microbiology, The Ohio State University, 484 West 12th Avenue, Columbus, OH 43210 (United States)
The E. coli transcriptional regulator RfaH was cloned, expressed, purified and crystallized and the complex of RfaH with its target DNA oligonucleotide was cocrystallized. Complete diffraction data sets were collected for the apo protein and its nucleic acid complex at 2.4 and at 1.6 Å resolution, respectively. 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{sub 1}22 or P6{sub 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 P1 (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.
- OSTI ID:
- 22356373
- Journal Information:
- Acta Crystallographica. Section F, Vol. 62, Issue Pt 10; Other Information: PMCID: PMC2225194; PMID: 17012804; PUBLISHER-ID: ll5089; OAI: oai:pubmedcentral.nih.gov:2225194; Copyright (c) International Union of Crystallography 2006; Country of input: International Atomic Energy Agency (IAEA); ISSN 1744-3091
- Country of Publication:
- United Kingdom
- Language:
- English
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