Physiocochemical and enzymatic studies of the bacteriophage T7 RNA polymerase-promoter complexes will be carried out using small restriction fragments of T7 DNA carrying functional promoters. Based on our previous sequence of the T7 RNA polymerase promoter the enzyme melts the sequence -A C T A T A G G - -T G A T A T C C -, conserved at each T7 promoter. We plan to determine the amino acid side chains involved in binding and melting the promoter and the base specificity of promoter recognition, melting and initiation, by selective base changes in the promoter sequence accomplished by synthetic or enzymatic means or by isolation of closely related but untranscribed promoters (i.e. T3 promoters). Methods will include chemical modification, limited proteolysis, and NMR (1H+31P) approaches to base structure at the promoter. Cloning of the gene 1 (T7 RNA polymerase, MW 98,000), gene 2.5 (SS-DNA binding protein, MW 25,000), gene 3 (endonuclease, MW 17,000), gene 4 (primase, MW 67,000) and gene 5 (T7 DNA polymerase, MW 81,000) into a plasmid containing a derepressible promoter is underway in an attempt to overproduce these gene products. This group of DNA binding proteins, involved in several aspects of DNA replication and transcription, provide a good experimental system for the study of solution structure and mechanism of proteins involved in the control of gene expression. Our ability to successfully attack the abnormalities that occur in gene function in genetic diseases and neoplasia will require a fundamental knowledge of the underlying enzymology involved in the regulation of gene expression. The gene 3 endonuclease will be developed as a probe of single-stranded or melted regions of DNA. As prototype DNA-protein complexes with oligonucleotides of defined sequence and high concentration accessible to multinuclear NMR methods, we are continuing our 1H, 19F and 31P studies of the structure of nucleotide complexes with helix-destabilizing proteins, gene 5 protein from fd, the E. coli SS-DNA binding protein and the T7 gene 2.5 DNA binding protein, the latter being acceleratory factors in DNA synthesis. The role of phosphorylatable proteins in the transcription of nucleosomal DNA is being investigated.
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