BRANCHED CHAIN AMINO ACID BIOSYNTHESIS IN E COLI

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DESCRIPTION: This proposal concerns the mechanism of transcriptional activation by a non-specific (histone-like) DNA binding protein called integration host factor (IHF), originally found and named based on its effect on site-specific integration of phage lambda and since shown to act also as a general regulator of expression of numerous operons. It is based upon the P.I.'s recent evidence that IHF activated the ilvG promotor of the ilvGMEDA operon for branched chain amino acid biosynthesis by a novel gene activation mechanism. He already showed that activation of the ilvG promoter requires binding of IHF to an upstream activation site (UAS1), a separate upstream site called SIDD, and a non cononical -10 region. He also showed that the IHF-binding site activated transcription when located on either face of the helix, thus making it unlikely that IHF interacts with RNAP or a different protein factor. He further substantiated this by substitution of the IHF binding site with the binding site of a heterologous DNA binding and bending protein, the lymphoid enhancer- binding factor LEF-1 activates the E. coli ilvG promoter. Binding and transcriptional activation under these conditions is accompanied by an unwinding of two A-T base pairs in the -10 promoter region. These and other experiments has led him to propose a "DNA structural-transmission model" for regulation of the ilvG promoter by IHF. His proposed new experiments are aimed towards testing a number of predictions of this model.