The goals of this proposal are to investigate the in vitro properties and in vivo function of TFII-I. TFII-1 is required for transcription initiation from many TATA-less promoters that instead contain an initiator element. This factor is also thought to provide for the integration of basal and regulatory transcription signals at these promoters. TFII-I contains 6 tandemly repeated HLH motifs that may mediate different protein/protein and protein/DNA interactions. The objectives of the proposal include detailed analysis of the functions of TFII-I employing DNA binding studies, binding site selection, in vitro transcription and in vitro mutagenesis. Determining the structure of TFII- I will be approached by Biochemical and Biophysical methods including high resolution crystallographic analysis. The proposal also includes investigations into the apparent regulation of TFII-I by phosphorylation in vivo and in vitro by phosphopeptide fingerprinting, in vitro mutagenesis and by employing dominant negatives of MAPK. The function of TFII-I at the V beta Inr element will be analyzed in vivo (in animal cells) using wild type and mutant TFII-I protein as well as V beta derived promoter constructs.
