The project is to investigate the molecular mechanisms of gene expression by using solution biophysical techniques to determine the structure-function relationships in DNA and RNA binding proteins involved in replication and transcription. These include T7 RNA polymerase, the ssDNA binding proteins, gene 32 from phage T4 (a 3-domain Zn(II) protein), gene 5 from phage M13, the hnRNA binding protein A1 from rat brain, and the retroviral ssDNA(RNA) binding proteins. All of these proteins, cloned in overproduction vectors, are available in the large quantities required for the following structural approaches. 2-D 1H-NMR, COSY and NOESY, are being applied to the smaller proteins and selectively to the larger proteins where nucleotide interactions perturb protons on specific AA residues. 1-D 1H-NMR is being combined with selective deuteration and site-directed mutagenesis to identify specific residues involved in nucleotide binding. Isolated protons contained in otherwise totally deuterated aromatic residues allow single 1H resonances to be resolved and used as probes in DNA-protein complexes as large as 300K. Other techniques for accessing protein-DNA complex formation will include fluorescent energy transfer, poly(d(A-T)) melting and domain isolation by limited proteolysis when possible. The synthesis of a 24 mer promoter on which a 5 base mRNA is made by T7 RNA polymerase will greatly aid kinetic studies of this enzyme and the study of the determinants for promoter complex formation by gel retention assays and by footprinting. Cloning of the two domains of the T7 RNA polymerase, a 20K N- term domain required for processivity and a C-term nonprocessive 80K domain containing the active center and DNA template binding site (which synthesizes the above 5 mer mRNA) will facilitate the study of small enzyme-promoter complexes by NMR and other biophysical techniques. Crystallization of all the above proteins is being actively pursued. Understanding the structure of proteins involved in gene expression appears to be our best hope of learning how to therapeutically interfere when disturbances in gene expression form the basis of pathological processes as in certain viral diseases and oncogenic transformations.
Showing the most recent 10 out of 35 publications
