In this renewal application we describe the progress of our research over the last four years on studies of the molecular mechanisms and controls that regulate the functions of DNA replication and RNA transcription complexes. Our studies have used primarily biophysical methods, and have primarily involved the replication complex of bacteriophage T4 and the transcription complex of E. coli. However, these systems feature essentially the same molecular mechanisms for 'driving' and regulating these central life processes as those that characterize 'higher organisms', including humans. As a result these studies provide good model systems to examine how human replication and transcription proceed at the fundamental level, and provide insights into what goes wrong at these levels in various forms of cancer and genetic diseases that often seem to involve minor kinetic or structural changes in the properties of these 'macromolecular machines'. During the last reporting period we completed a number of studies on the above mechanistic questions, using reconstituted replication or transcription complexes that carry out their functions with essentiall the same rates, fidelities and processivities as the in vivo versions of the same complexes within real cells. In these studies we placed fluorescent base analogue probes at defined positions within the nucleic acid frameworks of the reconstituted complexes, and then used fluorescent and circular dichroism spectroscopy at wavelengths great than 300 nm (an optical range in which the rest of the protein and nucleic acid components of the complexes are transparent) to monitor biologically relevant conformational changes at the probe sites. By these means we obtained significant information about replication and transcription mechanisms under steady state or equilibrium conditions. During the next reporting period we will follow up on preliminary studies that have shown that various versions of these same optical probe approaches can be used in more complex optical set-ups to permit two-dimensional fluorescence spectroscopic (2DFS) and single molecule Fluorescence Resonance Energy Transfer (smFRET) and Fluorescence Linear Dichroism (smFLD) measurements that can follow the kinetics of reactions within these complexes in 'real time' and with msec to msec resolution. This now gives us the opportunity to obtain local structural and dynamic information on changes at defined and biologically-relevant base analogue and DNA backbone probe sites, as well as to map transition states within individual rate-limiting molecular steps in transcription and replication. We believe that by using these approaches we can reveal new aspects of mechanisms and regulatory control systems that were previously inaccessible to direct experimental measurement, and should provide new and valuable fundamental information to understand cell processes and differentiation and to learn what goes wrong at the molecular level in related disease states.
Protein-nucleic acid complexes work in living cells to: (i) faithfully replicate (copy) the DNA genome to form the genetic material of the daughter cells; (ii) to transcribe and process specific messenger RNA sequences to permit 'expression' of the defined genes; and (iii) direct the synthesis of specific required proteins at various stages of development. These molecular complexes, which drive and direct these central processes of cellular function, largely self-assemble from individual protein and nucleic acid components to form the so-called 'macromolecular machines of gene expression'. We are continuing a program of research that uses the methods of physics and chemistry to understand the specific molecular processes that are involved in the functioning and regulation of these machines. Most of our work uses bacterial and viral systems as model organisms for simplicity, but the results also apply fully to our understanding of the processes of gene expression in higher organisms and humans. Understanding these central processes in molecular detail will help to develop specific tools to control diseases of anomalous gene expression and development, including various forms of cancer and related genetic diseases.
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