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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.

The Thermus thermophilus gene encoding the preprotein translocation ATPase SecA was cloned and expressed and the purified protein was crystallized by the hanging-drop vapour-diffusion technique in two different space groups P3(1(2))21 (a = b = 168.6, c = 149.8 A) and P6(1(5))22 (a = b = 130.9, c = 564.6 A). The crystals, improved by macroseeding, diffracted to beyond 2.8 and 3.5 A resolution for the trigonal and hexagonal crystal forms, respectively. Structure determination using the multiple isomorphous replacement method is in progress.

The Escherichia coli gene encoding the transcription cleavage factor GreB and the Thermus thermophilus gene encoding the anti-GreA transcription factor Gfh1 were cloned and expressed and the purified proteins were crystallized by the sitting-drop vapor-diffusion technique. The GreB and Gfh1 crystals, which were improved by macroseeding, belong to space group P4(1)2(1)2 (or P4(3)2(1)2), with unit-cell parameters a = b = 148, c = 115.2 A and a = b = 59.3, c = 218.9 A, respectively. Complete diffraction data sets were collected for the GreB and Gfh1 crystals to 2.6 and 2.8 A resolution, respectively. Crystals of the selenomethionine proteins were obtained by microseeding using the native protein crystals and diffract as well as the native ones. The structure determination of these proteins is now in progress.

The Escherichia coli gene encoding a regulator of stringent response and virulence, DksA, which contains a canonical Zn finger motif, was cloned and expressed, and the purified protein was crystallized by the hanging-drop vapor-diffusion technique in two different space groups, P2(1)2(1)2(1) (a = 91.32, b = 96.59, c = 117.48 A) and C222 (a = 80.6, b = 115.1, c = 149.57 A). The crystals belonging to space group P2(1)2(1)2(1), improved by macroseeding, diffract beyond 2.0 A at a synchrotron. Three complete atomic resolution multiple anomalous dispersion diffraction data sets were collected from the same crystal of the P2(1)2(1)2(1) crystal form at the absorption edge for Zn atoms.

The gene encoding LysX, an essential component of the lysine-biosynthesis pathway in Thermus thermophilus (molecular weight approximately equal 31,000 Da), was cloned and expressed and the purified protein was crystallized by the hanging-drop vapour-diffusion technique in two different space groups, C2 (unit-cell parameters a = 124.7, b = 51.4, c = 103.6 A, beta = 122.8 degrees ) and R3 (a = b = 122.6, c = 97.6 A). Crystals improved by macroseeding diffracted to beyond 2.3 and 3 A resolution for the C2 and R3 crystal forms, respectively. Complete diffraction data sets were collected for the C2 and R3 crystal forms at 2.5 and 3.1 A resolution, respectively. Crystals of selenomethionine-containing LysX protein were obtained by cross-microseeding, using the native microcrystals as a seed. Structure determination is now in progress.

RNA polymerase holoenzyme from Thermus thermophilus, consisting of six protein subunits (alpha(2), beta, beta', omega and sigma(70)) and having a total molecular mass of about 450 kDa, was purified and crystallized by the hanging-drop vapour-diffusion technique under mild near-physiological conditions. The crystals diffract beyond 3 A resolution. Careful analysis of diffraction data revealed that the crystals belong to space group P3(2), with unit-cell parameters a = b = 236.35, c = 249.04 A, and have perfect twinning along the threefold axis. A complete data set at 3 A resolution was collected and an unambiguous molecular-replacement solution was found using the structure of T. aquaticus RNA polymerase core enzyme as a search model. The refinement of structure and model building of the sigma(70) subunit is now in progress.