EXPRESSED PROTEIN LIGATION, A NOVEL METHOD FOR STUDYING PROTEIN-PROTEIN INTERACTIONS IN TRANSCRIPTION

Expressed protein ligation is a novel protein semisynthesis method tha t permits the in vitro ligation of a chemically synthesized C-terminal segment of a protein to a recombinant N-terminal segment fused throug h its C terminus to an intein protein splicing element. In principle, the practical convenience of this method, combined with the expanded o pportunities in protein engineering that it provides, makes it well su ited for probing the molecular basis of complex processes such as tran scription. Here we describe the successful application of expressed pr otein ligation to the similar to 600 amino acid sigma(70) subunit of E scherichia coil RNA polymerase. The resulting semi-synthetic sigma(70) constructs are shown to be fully functional and have been used to map the binding region of the bacteriophage T4 anti-sigma protein, AsiA, to within amino acids 567-600 of sigma(70). The success of these semi- synthesis studies sets the stage for the future generation of semi-syn thetic sigma(70) molecules in which unnatural amino acids and biophysi cal probes are site-specifically incorporated in the RNA polymerase co mplex.