 Steenbock Professor of Chemical Sciences John D. Ferry Professor of Chemistry and Biochemistry, Born 1942 B.A. 1964, Yale University Ph.D. 1967, University of California, San Diego Room: 3204B Biochem SciencesPhone: 608-262-5332 Email: record@chem.wisc.edu Position: Professor Selected Publications
| Research DescriptionProtein-nucleic acid interactions, including kinetics and mechanism of transcription initiation, characterization of DNA wrapping in protein DNA complexes, development of small molecule solutes as thermodynamic and mechanistic probes of protein and DNA conformational changes. Protein-Nucleic Acid Interactions (PNAI) Protein-DNA interactions are central to all DNA processes, including the storage, replication, and expression of genetic information in the cell. The Record lab has pioneered the use of quantitative physical biochemical approaches to describe these interactions both experimentally and theoretically. Current PNAI projects in the lab involve characterization of the assembly and function of: E. coli RNA polymerase-promoter DNA open complexes in transcription initiation; and wrapped/bent DNA complexes formed with the histone-like proteins Integration Host Factor and HU. All these systems are unified by a common theme: large conformational changes and other coupled processes in the proteins and/or their target DNA sites occur in binding. To study these processes, we use chemical and enzymatic DNA footprinting, microcalorimetry, circular dichroism, fluorescence, nitrocellulose filter binding, rapid mix-quench kinetics, and structural modeling. 1. RNAP-Promoter Open Complex Formation. How and when is DNA opened in the initial steps of transcription initiation? What roles do the different domains of polymerase play during this highly regulated process? We are using fast footprinting and rapid quench methods to answer these questions; current results summarized in the figures below. Biophysical studies of protein-nucleic acid interactions in transcription initiation in vitroand in E. coli,computational and experimental studies of polyelectrolyte properties of nucleic acids and their complexes.
Model of I1: Wrapping of upstream DNA around RNAP opens the downstream jaw, allowing the DNA containing the ranscription start site to enter the active site channel. Davis et al. PNAS 104: 7833-38, 2007
Kontur et al. Biochemistry 45: 2161-2177, 2006
2. IHF/HU-DNA interactions: model systems for the study of coupled folding and DNA wrapping. These two structurally homologous proteins play key roles in organizing the bacterial nucleoid and are involved in numerous DNA transactions (transcription, replication, repair). To gain insight into their functions, we are studying their specific and nonspecific binding modes and how these modes are influenced by solution variables.
34 bp mode 10 bp mode Model of the transition from a 34 bp DNA binding mode to 10 bp mode as the molar ratio of HU to DNA increases. The shift to a smaller binding mode pulls the beta arms of HU out of the DNA minor groove. The U-shaped DNA bend is lost with the removal of the two proline “levers” (green). Strikingly, the two modes have a similar number of contacts between positively charged side chains (blue) with negatively charged DNA phosphate oxygens (red), consistent with the observation that changes in salt concentration affects the binding affinity of both modes to the same extent. (Koh et al., in preparation, 2009). Structural Interpretation and Predictions of the Effects of Solutes and Hofmeister Salt Ions on Biopolymer Processes Interactions of solutes and salts with biopolymer surfaces play a large role in determining the kinetics and thermodynamics of biopolymer processes such as folding, assembling, binding and crystallization. Recent research in the Record lab has focused on the development of a model and molecular thermodynamic analysis to interpret and predict these effects quantitatively in terms of structural information. This can be done using the Solute Partitioning Model (SPM), a two-state model that interprets the effects of a solute on a biopolymer surface in terms of the change in water accessible surface of the biopolymer and a partition coefficient Kp quantifying the local concentration of the solute in the water of hydration of that surface, relative to its bulk concentration. A database of partition coefficients for salt ions and solutes is being established from analysis of experiments with biopolymers and model compounds. With this database and structural information about the interface formed in a protein-protein or protein-nucleic acid complex, for the first time one will be able to predict the effect of a solute or Hofmeister salt on the thermodynamics and kinetics of protein and nucleic acid processes, and interpret differences between predicted and observed solute effects in terms of coupled processes such as large-scale conformational changes coupled to binding.
The salt-ion partitioning model superimposed over an idealized representation of molecular dynamics simulation results showing exclusion of Na2SO4 from the air–water interface. Pegram and Record, Chem. Phys. Lett. 467, 1-8 (2008). Awards
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