In this competing continuing research grant application we propose to extend our ongoing studies on the basic principles of protein- nucleic acid interactions that underlie the regulation of gene expression. During this reporting period we plan to focus on an analysis of the E. coli RNA polymerase transcription complex, and will use thermodynamic, kinetic and crosslinking methods to analyze the structure and function of the steady-state transcription elongation complex, the nature of pausing in this complex, and the relation of this pausing to """"""""factor-independent"""""""" and rho-dependent transcription termination. In these studies we will isolate and study specifically paused and stable lambdal and T7 phage elongation complexes. We will also use these complexes to examine the binding of various regulatory proteins (including sigma factor, nusA protein, rho protein, etc.) to the core polymerase of the elongation complex. In addition we will continue our studies of the mechanisms of transcript release by rho protein at rho-dependent transcript termination sites. Physico-chemical studies of the mechanisms of rho protein oligomerization, of RNA and ATP binding to rho. and of the RNA-dependent activation of rho ATPase will also be continued. Finally, we will continue to develop general theoretical and experimental methods for the study of protein-nucleic acid interactions. As before, we will attempt both to examine specific physiologically-active protein complexes involved in gene expression, and to elucidate some of the general principles that underlie the function of all such systems.
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