Regulatory mutants which affect attenuation of the thr operon of Escherichia coli will be constructed by oligonucleotide-directed mutagenesis and by direct synthesis procedures. We will introduce frameshift mutations into the leader peptide coding sequence to determine which region(s) are critical for regulation. We will also construct substitution mutations which affect RNA secondary structures that are thought to be important for regulation. In addition, we will study the effects DNA superhelix density and termination factors on transcription termination in vitro. The long range goal of this work is to understand the molecular events that regulate transcription termination at the thr attenuator. A second project is concerned with the molecular mechanism of bacteriophage lambda site-specific recombination. Mutations will be introduced in vitro into the binding sites where recombination proteins bind during recombination. The mutations will be characterized by their effects on recombination and by their interactions with recombination proteins in DNA protection assays. We will also construct novel DNA substrates to study the roles of branch migration and DNA-DNA interactions during recombination. The long range goal of this research is to understand the molecular mechanism of strand exchange and to gain information on the organization and assembly of recombination complexes.
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