In this work we will exploit the preliminary observation that non-denaturing polyacrylamide gel electrophoresis is able to separate discrete intermediates in the pathway of transcription initiation. These have been tentatively identified as """"""""closed"""""""" and """"""""open"""""""" polymerase-promoter complexes, and an """"""""initiated"""""""" complex containing a stably bound nascent RNA. The objectives include 1) further confirmation of the complex identities, 2) characterization of equilibria and kinetics for conversion among the states, and 3) use of enzymatic and chemical footprinting methods to identify the protein-DNA contact regions. A new method, called functional domain mapping, will be used to discover what region of the DNA sequence is required for formation of a particular polymerase-promoter complex which is stable enough to withstand gel electrophoresis. In addition, 4) we will search for DNA conformational modification effects, such as systematic bending or increased flexibility, in the discrete initiation intermediates, 5) examine the complexes for protein structural changes, such as loss of particular subunits, (especially sigma), and changes in protein-protein contacts as revealed by chemical crosslinking, and 6) examine ternary complexes formed in the presence of lac repressor or the cAMP-binding gene activating protein CAP. These latter experiments will seek to elucidate the effect of gene regulatory proteins on the steps of transcription initiation, through study of their influence on kinetics and equilibria of the reaction steps, and on conformational influences such as DNA bending. Protein-protein contacts in discrete ternary complexes will be investigated by chemical crosslinking methods. Finally, 7) we will study the rate and mechanism of conversion from a complex undergoing abortive initiation to the complex with stably bound RNA. Additional aims shared with other projects include study of the effect of DNA-binding antitumor drugs on the stages and intermediates in transcription initiation.
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