Ronald Somerville

Professor of Biochemistry

• About
• Selected Publications

	Area	Molecular Biology of the TrpR Protein
	Email	somerville@purdue.edu
	Degree	Ph.D. University of Michigan, 1961
	Retired	2004

Molecular Biology of the TrpR Protein

The formation of stable noncovalent complexes between regulatory proteins and punctuation elements in duplex DNA is central to the expression of controlled genes. A suitable model system for studying the fundamentals of protein-DNA interaction is TrpR protein of Escherichia coli. This protein is a homodimer (monomer MW 12,356) whose primary, secondary and tertiary structures are known, and whose gene (trpR) is available in a variety of useful vectors. Ordinarily TrpR protein has weak affinity for DNA; however, when L-tryptophan binds to the protein, a conformational change occurs. The resulting liganded species has high affinity for several operator targets, many of which lie within promoters specific for genes related to the biosynthesis of tryptophan. Using wild-type and mutant TrpR proteins, we are analyzing the physicochemical parameters that govern monomer-dimer equilibrium, ligand binding and operator recognition. Using oligonucleotide-directed mutagenesis, we have generated a large collection of mutant proteins having single amino acid switches within a specific segment of TrpR (the so-called "D" helix) whose role in protein-DNA binding remains enigmatic. The effects of these switches on various properties of the protein are being studied in vivo and in vitro.E. coli cytoplasm contains several proteins that can interact with TrpR. The identity of these proteins and their functional roles are being pursued using biochemical, genetic, and immunochemical approaches.

Transcriptional Regulation by the TyrR Protein

The TyrR protein of E. coli is a homodimer having 513 amino acids per subunit. In concert with phenylalanine and tyrosine, this factor regulates transcription from at least eight promoters. Transcriptional regulation by the TyrR protein is complex and interesting, in that certain promoters (e.g. aroF) are sharply inhibited by the binding of a TyrR/ligand complex to an operator target, while other promoters (e.g. mtr,tpl) are stimulated by similar protein-DNA interactions. A third pattern of regulatory response is seen for the tyrP promoter, which is repressed when tyrosine is the ligand and induced when phenylalanine is supplied. To clarify how the TyrR protein mediates such diverse regulatory effects, we are conducting a series of biochemical, genetic, and structural studies aimed at 

1. describing the domain structure of the TyrR protein
2. localizing sites for the binding of aromatic amino acids and ATP
3. identifying polypeptide segments important in the establishment and maintenance of the dimeric state
4. identifying polypeptide segments important in the activation of transcription
5. defining in mechanistic terms how the binding of TyrR protein near certain promoters enhances the frequency of transcription initiation.

Recently we have begun structure-function studies of the TyrR protein of Haemophilus influenzae (318 amino acid residues).  This protein has the same operator recognition characteristics as its E. coli counterpart but is unable to activate transcription from mtr or tpl.  Preliminary results suggest that the H. influenzae TyrR protein may be susceptible to structural analysis by high resolution NMR.