Browsing by Author "Augustus, Anne Marie"

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    Interactions of the MetJ Repressor from E. Coli with DNA and SAM
    (2009) Augustus, Anne Marie

    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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    The Chloroplast Tubulin Homologs FtsZA and FtsZB from the Red Alga Galdieria sulphuraria Co-assemble into Dynamic Filaments.
    (J Biol Chem, 2017-02-07) Chen, Yaodong; Porter, Katie; Osawa, Masaki; Augustus, Anne Marie; Milam, Sara L; Joshi, Chandra; Osteryoung, Katherine W; Erickson, Harold P
    FtsZ is a homolog of eukaryotic tubulin and is present in almost all bacteria and many archaea, where it is the major cytoskeletal protein in the Z ring, required for cell division. Unlike some other cell organelles of prokaryotic origin, chloroplasts have retained FtsZ as an essential component of the division machinery. However, chloroplast FtsZs have been challenging to study because they are difficult to express and purify. To this end, we have used a FATT-tag expression system to produce as soluble proteins the two chloroplast FtsZs from Galdieria sulphuraria, a thermophilic red alga. GsFtsZA and GsFtsZB assembled individually in the presence of GTP, forming large bundles of protofilaments. GsFtsZA also assembled in the presence of GDP, the first member of the FtsZ/tubulin superfamily to do so. Mixtures of GsFtsZA and GsFtsZB assembled protofilament bundles and hydrolyzed GTP at a rate approximately equal to the sum of their individual rates, suggesting a random co-assembly. GsFtsZA assembly by itself in limiting GTP gave polymers that remained stable for a prolonged time. However, when GsFtsZB was added, the co-polymers disassembled with enhanced kinetics, suggesting that the GsFtsZB regulates and enhances disassembly dynamics. GsFtsZA-mts (where mts is a membrane-targeting amphipathic helix) formed Z ring-like helices when expressed in E. coli. Co-expression of GsFtsZB (without an mts) gave co-assembly of both into similar helices. In summary, we provide biochemical evidence that GsFtsZA assembles as the primary scaffold of the chloroplast Z ring, and that GsFtsZB co-assembly enhances polymer disassembly and dynamics.