In this grant application we describe plans to continue our ongoing studies of the molecular mechanisms and regulation of the transcription complex of E. coli. We will focus, in particular, on how the complex is regulated by transcription factors, building on the recent major progress that has greatly increased our understanding both of many aspects of the structure of the complex, as well as of the thermodynamics and kinetics of the control of the reactions that lead to transcript initiation, elongation, editing, and termination. This later progress, in particular, makes it possible for us to begin to develop quantitative insight into the changes that the binding of transcription factors must bring about to (e.g.) redirect the transcription process from elongation to transcript editing or termination. During the next reporting period our Specific Aims will be: (i) to continue our studies of the mechanisms whereby the N protein-dependent antitermination system of phage lambda controls the transcription termination efficiency of E. coli RNA polymerase at intrinsic and rho-dependent terminators; (ii) to carry out fundamental studies of RNA flexibility and looping as a component of transcription regulation; (iii) to elucidate the role of specific RNA sequences and E. coli host factors in regulating the """"""""range"""""""" and specificity of """"""""full"""""""" transcription factor complexes involved in N-dependent antitermination; (iv) to determine the detailed mechanism of action of E. coli transcription termination factor rho at rho-dependent terminators, and to study the roles of """"""""coupling factors"""""""" in regulating the efficiency of the rho-dependent termination process; (v) to examine the interconversion of the various forms of the E. coli transcription elongation complex that lead, respectively, to elongation, editing, and termination, as well as to use rho as a probe of these """"""""functional states""""""""; and (vi) to perform theoretical and modeling studies of the assembly and stability of functional complexes of transcription factors to better understand how these complexes assemble, and how the components interact to build a stable """"""""macromolecular machine"""""""". In terms of their significance for biomedical research, these studies will serve as molecular models for the function and control of the analogous DNA-dependent RNA transcription systems of higher organisms, and may help reveal how these controls can go awry in cancer and other diseases of inappropriate gene expression.
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