In this renewal application, we describe plans to continue and complete several ongoing studies on the mechanisms and control of the transcription complex of E. coli and the replication complex of phage T4. Both represent the simplest versions of the equivalent 'macromolecular machines' of gene expression that use most of the same components to control these processes in higher organisms. We will focus, in particular, on how the central polymerases perform the single nucleotide addition cycle in template-directed synthesis, and how this process is controlled and redirected into alternative and less probable kinetically competing reaction pathways (including termination and editing) at pause sites, at termination sites, and as a consequence of residue mis-incorporation. We will also focus on how these 'primary' reaction pathways are further modulated by regulatory factors, such as anti-termination complexes. In support of these overall objectives, our Specific Aims during the next reporting period will be to further examine the following issues: (i) to develop and exploit a new spectroscopic method for studying local RNA and DMA conformations and dynamics; (ii) to further examine the steps in the single nucleotide addition cycle in transcription and replication; (iii) to continue to study the control of reaction pathway selection in transcription; (iv) to further understand the basic control mechanisms of antitermination systems; (v) to complete our model studies of RNA and protein chain looping and dynamics; (vi) to examine the mechanistic details of the interactions of E. coli transcription termination factor Rho with RNA polymerase; (vii) to continue our studies of helicase mechanisms and coupling in transcription and replication; (viii) to further examine the mechanisms of processivity clamp loading in the phage T4 system; and (ix) to continue our theoretical studies of protein-nucleic acid interaction kinetics. These studies should help us to better understand how these complexes assemble, and how the components interact to build stable, and yet easily regulatable 'macromolecular machines'. In terms of their significance for biomedical research, these studies will continue to serve as models for the function and control of the analogous transcription and replication systems of higher organisms, and may help reveal how these complexes can go awry in cancer and other diseases of inappropriate gene expression. ? ?
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