9722371 Kahn DNA transactions such as replication, recombination, and transcription are all performed by large, organized multi-protein DNA complexes. The three-dimensional pathway of the DNA and the arrangement of proteins within these higher-order complexes are fundamental to their functions. The higher-order complexes that mediate gene regulation often include "architectural" proteins that bend DNA, thereby apposing other factors which otherwise would be separated, and presumably changing their interactions with each other or with RNA polymerase. Understanding these combinatorial patterns of regulation is of great interest, especially as knowledge of the structures of the critical multi-protein DNA intermediates will be essential for controlling their functions, with attendant biomedical implications. This research will focus on putative DNA wrapping around E. coli RNA polymerase during transcription initiation and elongation. This wrapping may be mechanistically important and may have consequences for the large-scale organization of DNA in the cell. The existing methods for probing this large-scale structure, including biochemical methods, gel electrophoresis, and microscopy, all have limitations. A new methodology will be developed based on the enhanced cyclization or ring-closure of DNA whose ends have been brought together and aligned by bending or wrapping and on selection-amplification methods based on ligation of DNA fragments to form minicircles. The starting population will be a large collection of DNA molecules bearing the specific DNA sites of interest and a segment with random length and curvature, synthesized by ligation of a mixture of curved and straight oligonucleotides of known solution structure. Molecules which cyclize rapidly will be selected and amplified using the polymerase chain reaction, and the process repeated. The selected DNA molecules will be cloned and sequenced, and based on known properties of the components used in the random synthesis and on kine tic measurements of DNA cyclization, three-dimensional models for the higher-order complex will be obtained. This procedure will be applied to the E. coil RNA polymerase DNA open complex, to complexes including the CAP protein, and to later intermediates in the transcription cycle. This work will be pursued in the context of an integrated career, involving undergraduates as well as graduate students in the research. Teaching in undergraduate and graduate courses will be pursued concurrently. Computer-based methods for involving students in the course work and for encouraging them to explore on their own will be developed. A departmental hardware and software resource center for teaching undergraduate and graduate biochemistry also will be created. %%% DNA transactions such as replication, recombination, and transcription are all performed by large, organized multi-protein DNA complexes. The three-dimensional pathway of the DNA and the arrangement of proteins within these higher-order complexes are fundamental to their functions. This research is specifically directed toward understanding large scale structures involved in gene expression and will be pursued in the context of an integrated career, involving undergraduates as well as graduate students in the research. Computer-based methods for involving students in course work and for encouraging them to explore on their own as well as a departmental hardware and software resource center for teaching undergraduate and graduate biochemistry will be developed. ***
