In this renewal application we describe progress over the past four years, together with our plans to continue and complete several ongoing studies dealing with the mechanisms and control of the RNA transcription complex of E. coli and the DNA replication complex of bacteriophage T4. Both systems can be viewed as 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, and as such these studies will help to elucidate general basic mechanisms and may also help to reveal how such complexes and their controls go awry in cancer and other diseases of inappropriate gene expression. Our focus is on how the central polymerases of these machines carry out the single nucleotide addition cycle in template-directed synthesis, and how this process is controlled and redirected into alternative and less probable reaction pathways that compete with elongation at pause sites, at termination sites, and as a consequence of base misincorporation. We also focus how these """"""""primary"""""""" reaction pathways are further modulated by regulatory factors that interact with these complexes. Our Overall Research Goals and the Specific Aims we plan to accomplish in implementing these goals during the next reporting period are as follows. I. To determine the molecular and structural mechanisms of transcript elongation and of the competing reaction pathways that represent alternatives to elongation.
Specific Aim 1. To define the thermodynamic parameters of the chemical steps of the single nucleotide addition cycle in transcription (and replication) and the control of polymerase pausing.
Specific Aim 2. To use SPR and gel measurements on defined templates to determine the dependence of the kinetic parameters of the transcription elongation reaction on template sequence and transcription factor binding.
Specific Aim 3. To determine the detailed mechanisms of E. coli factor Rho functioning as a RNA-DNA helicase to terminate transcription. II. To elucidate the general principles of helicase function and coupling within macromolecular machines and their relation to DNA dynamics and structure.
Specific Aim 4. To define the relationship between DNA """"""""breathing"""""""" and helicase action using the T4 replication helicase (primosome) complex.
Specific Aim 5. To develop and test new spectroscopic base-analogue probes for steady-state and kinetic studies of conformational changes in macromolecular machines.
Specific Aim 6. To investigate stacking between nucleic acid base analogue probes and functionally important aromatic amino acid residues of genome regulatory proteins.
Specific Aim 7. To apply (and develop) new spectroscopic methods to study the dynamics of conformational changes within functioning macromolecular machines in the ?sec to sec time range with initial applications to DNA base-pair breathing at replication forks (with and without helicase) and to the shuttling of primer strand between the pol- and exo- active sites in DNA polymerase.
Specific Aim 8. To determine the role of conformational change in template bases in controlling DNA replication fidelity. III. To understand the molecular details and control of processivity clamp loading and helicase-primase integration with polymerase within the overall T4 DNA replication complex.
Specific Aim 9. To complete mechanistic studies of the processivity clamp loading process in the T4 DNA replication system, including further study of the role of ATP in the loading process and the interaction of the clamp with the P/T DNA junction and the replication polymerase.
Specific Aim 1 0. To establish the assembly pathway of the helicase-primase complex of the T4 bacteriophage T4 replication system and define the steps involved in integrating primase and helicase functions.
Protein and 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) transcribe and process specific messenger RNA sequences to bring about expression of defined genes;and (iii) direct the synthesis of specific required proteins in the 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 physical biochemical methods to understand the specific molecular steps that are involved in function and control of these machines. We focus on the RNA transcription complex of the bacterium E. coli, and the DNA replication complex of the T4 bacterial virus. Our results are helping to reveal how these complexes work in molecular detail and providing information to identify specific interaction targets within the equivalent machines of higher organisms. Such targets can then be exploited to develop specific tools and reagents to control diseases of anomalous cellular development, including various forms of cancer and other genetic diseases.
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