Structure of Bacteriophage T7 Rna Polymerase at 2.6 Å Resolution and a New Low-Salt Crystal Form. B.-C. Wang¹, Z.-J. Liu¹, C.-J. Chen^1,2, K. Chandrasekhar¹, W.-R. Chang¹, T.-G. Huang¹, Z.-L. Wan¹, M.-Y. Liu¹, R. Sousa³, and J. P. Rose¹, ¹Department of Biochemistry and Molecular Biology, University of Georgia, Athens, GA 30602, ²Department of Crystallography, University of Pittsburgh, Pittsburgh, PA 15260, and ³Department of Biochemistry, University of Texas Health Science Center, San Antonio, TX 78284

The resolution of a high-salt crystal structure of bacteriophage T7 RNA polymerase (T7 RNAP) has now been extended from 3.3 Å (Sousa et al, Nature, 364, 593-599, 1993) to 2.6 Å using data collected at -186 deg.C. The high-salt crystal form belongs to space group P2₁ with cell dimensions (low temperature) a = 112.6 Å, b = 137.8 Å, c = 122.7 Å, and b = 96.6deg.. There are 3 molecules (882 residues each) per asymmetric unit related to each other by a local 3₁ symmetry. The phase extension process included re-refinement of heavy atom sites, density modification, and 6-fold density averaging between the room-temperature and the low-temperature crystals using 'DM'. The new map confirmed the folding observed from the original 3.3Å map for the C-terminal and the central regions, and resolved the ambiguity in the N-terminal region of the molecule. The current model (3 molecules) contains 2,280 residues (86% complete). Other residues may be disordered. Unrefined Ca-coordinates have been deposited in the Protein Data Bank (entry 3RNP). Structural refinement is in progress.

A low-salt crystal form of T7 RNAP has been obtained (grown by C.-J.C). These crystals belong to space group P3₁21, have one molecule per asymmetric unit, and diffract to at least 3.0 Å resolution using a rotation anode. The crystal structure has been determined by molecular replacement using the high-salt structure as a search model. A preliminary comparison shows that the two structures are very similar in these two crystal forms. In addition, the molecular packing along the local 3₁ symmetry in the P2₁ crystals is preserved in the P3₁21 crystals, running along the crystallographic 3₁ axis.

Work supported by NIH Grant RO1 GM41936 and the University of Georgia Research Foundation.