Vassylyeva Marina N, Svetlov Vladimir, Klyuyev Sergiy, Devedjiev Yancho D, Artsimovitch Irina, Vassylyev Dmitry G
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.
Acta Crystallogr Sect F Struct Biol Cryst Commun. 2006 Oct 1;62(Pt 10):1027-30. doi: 10.1107/S174430910603658X. Epub 2006 Sep 30.
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.
细菌转录因子及毒力调节因子RfaH通过与非模板DNA链的暴露片段进行特异性相互作用，结合到快速移动的转录延伸复合物上。为阐明这种不同寻常的募集机制，我们开始测定RfaH及其与DNA复合物的三维结构。为此，克隆并表达了大肠杆菌的rfaH基因。通过坐滴气相扩散技术使纯化后的蛋白结晶。空间群为P6(1)22或P6(5)22，晶胞参数a = b = 45.46，c = 599.93 Å。采用相同技术，但在不同结晶条件下，使RfaH与一个九核苷酸寡脱氧核糖核苷酸的复合物结晶，得到属于空间群P1的晶体（晶胞参数a = 36.79，b = 44.01，c = 62.37 Å，α = 80.62，β = 75.37，γ = 75.41°）。分别在2.4 Å和1.6 Å分辨率下收集了RfaH及其与DNA复合物的完整衍射数据集。通过使用天然微晶进行交叉微种晶，获得了两种晶体形式的硒代甲硫氨酸标记蛋白的晶体。RfaH及其与DNA复合物的结构测定正在进行中。
