Protein-Nucleic Acid Interactions
The ring-forming oligomeric Bacillus trp RNA binding attenuation protein (TRAP), defines a paradigm for gene regulation by ligand-mediated alteration of the structure of non-coding RNA, and for mechanisms of both homotropic and heterotropic allostery. Undecameric (11-mer) TRAP serves as a sensor for intracellular tryptophan (Trp), which occupy its 11 identical sites, and thereby activates the protein for binding to specific RNA sequences in the 5′ untranslated regions of messenger RNAs. RNA binding by activated TRAP results in remodeling of RNA secondary structures that include (1) competing hairpins whose structure regulates transcription via aborted transcripts (termination), and (2) competing hairpins that regulate translation by alternately exposing or sequestering the ribosome binding site. The repressive RNA binding activity of Trp-activated TRAP can be blocked by the binding of another oligomeric protein, Anti-TRAP (AT), whose biosynthesis is regulated by sensing of the levels of uncharged tRNATrp.
During protein translation, aminoacyl-tRNA synthetases (aaRSs) are responsible for covalently attaching amino acids to cognate tRNAs in a process knownas aminoacylation. High fidelity in this step is critical to ensure that incorrect amino acids are not incorporated into proteins. Prolyl-tRNA synthetase (ProRS) is mischarges tRNAPro with both alanine and cysteine, while INS superfamily of proteins are responsible for hydrolyzing these mischarged tRNAs in all three domains of life. ProXp-ala and YbaK are two such proteins, responsible for clearing Ala-tRNAPro and Cys-tRNAPro, respectively. In collaboration with Karin Musier-Forsyth’s lab, we are utilizing NMR and a combination of biochemical and biophysical techniques to better characterize these editing complexes.
The Loz1 transcription factor from Schizosaccharomyces pombe plays an essential role in zinc homeostasis by repressing target gene expression in zinc-replete cells. Biochemical and genetic experiments performed in the laboratory of Amanda Bird indicate that a 96-amino acid C-terminal region of the 522-residue protein, containing a double C2H2 zinc finger domain and an accessory domain that enhances DNA binding, are necessary and sufficient for DNA binding and zinc-dependent repression. We are using NMR spectroscopy to determine the structural basis for Zn-dependent gene regulation mediated by Loz1.
RNase P is an essential ribonucleoprotein enzyme found in all living organisms. The RNA subunit is catalytic although the protein(s) are required for in vivo activity. The composition of RNA and proteins from RNase P varies among organisms; simple organisms tend to have larger RNA and fewer protein subunits while RNase P in more complex organisms is composed of a smaller RNA but a larger number of protein subunits. We collaborate with the laboratory of Venkat Gopalan is to understand the mechanisms by which the protein subunits modulate the activity of the catalytic RNA by studying the structure and interactions of those components in model archaeal organisms, including Pyrococcus furiosus (Pfu).
Dynamics in Tyrosine Recombinases
Cre is a member of a large family of phage-derived enzymes known as tyrosine recombinases (e.g., λ-integrase, Flp recombinase), which play important functions in viral infection, gene transposition, and bacterial pathogenesis. Cre has become a widely-used genetic engineering tool for two powerful applications: restriction enzyme-free DNA cloning and conditional expression of target genes. Despite its widespread use in biotechnology and many high-resolution crystal structures of Cre-DNA complexes (23 coordinate sets in http://rcsb.org, with resolutions down to 2 Å), fundamental gaps exist in our understanding of the mechanism of site selection, coordinated DNA strand recognition, cleavage, exchange, and re-ligation. We are using isotope labeling and TROSY NMR of lambda and Cre recombinases to illuminate the roles of protein dynamics in the function of these enzymes.