Interactions of the MetJ Repressor from E. Coli with DNA and SAM
Transcription regulators are proteins that bind to specific DNA sequences in order to control the expression of specific genes. Often the sequences that are bound are not identical, but contain deviations from a common "consensus" sequence. The proteins that recognize these non-consensus sites must be able to recognize a variety of related sequences. MetJ is the transcription regulator that controls the expression of genes involved in methionine biosynthesis and transport in E. coli and other related organisms. A consensus sequence is known, but almost all the naturally occurring binding sites for MetJ differ from this. The goal of this dissertation is to understand how MetJ recognizes its various target sites within the context of the genomic DNA in which they are embedded. This work uses a variety of biochemical and biophysical techniques to further our understanding of an important regulatory protein.
Chapter 2 describes the results of both in cell and in vitro NMR showing that MetJ associates with non-specific genomic DNA in the cell, and that specific DNA (containing the consensus sequence) can successfully compete with a large excess of non-specific DNA for MetJ binding.
Chapter 3 describes work performed with small-angle neutron scattering showing different modes of MetJ binding to DNA of variable length and sequence.
Chapter 4 extends the neutron-scattering results by using analytical ultracentrifugation to look at MetJ binding to a wide variety of DNA sequences, both in the presence and absence of its co-factor, S- adenosylmethionine (SAM). Evidence is presented for SAM-mediated binding to both specific and non-specific DNA, as well as the importance of cooperativity in binding multiple MetJ molecules to a single DNA.
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