The objective of this research is to provide, through crystallographic studies, information which will help to understand the structure-function relationship of RNA polymerase at the atomic level. A large amount of T7 RNA polymerase has recently been isolated from an overexpression system, and experimental conditions have been established under which diffraction quality single crystals can be grown. The crystals, which belong to space group P21 with cell constants a = 114.5, b = 138.6, c = 125.7 angstrom, and beta = 98.1 degree, grow to a size of 1.5 x 0.2 x 0.1 mm, and diffract to at least 3.5 angstrom resolution. A systematic crystallographic study is proposed on a series of RNA polymerases and their DNA complexes.
Specific aims are as follows: (1) to determine the crystal structure of the T7 RNA polymerase crystals presently in hand, and to extend the diffraction resolution of these crystals and analyze their structural details, using synchrotron radiation data. (2) to grow and study the T7 RNA polymerase-DNA co-crystals containing the class II and class III T7 promoters and nucleoside triphosphates. (3) to grow native crystals of bacteriophage T3 RNA polymerase, and its DNA complexes, with the aim of solving their crystal structures. Success in obtaining diffraction quality single crystals of T7 RNA polymerase represents the first significant step toward a complete crystallographic study of the enzyme. The proposed research is expected to provide new information and knowledge necessary for the understanding of the structure-function relationship of RNA polymerase and its involvement in DNA transcription.
