Browsing Theses and Dissertations by Author "Paudyal, Samridhdi"
FUNCTIONAL ANALYSIS OF THE BACTERIAL MACRODOMAIN PROTEIN YMDB AND ITS INTERACTION WITH RIBONUCLEASE IIINicholson, Allen W.; Waring, Richard B.; Balciunas, Darius; Voelz, Vincent (Temple University. Libraries, 2014)The Escherichia coli ymdB gene encodes a ~19 kDa protein that binds ADP-ribose (ADPR) and metabolites related to NAD+. As such, it has been termed a macrodomain protein, referring to a conserved fold that binds ADPR. YmdB can catalyze the hydrolysis of O-acetyl-ADP-ribose (OAADPR), forming acetate and ADPR. OAADPR is a product of sirtuin action on lysine-acetylated proteins, which involves NAD+ as a cosubstrate. There is evidence that YmdB interacts with other proteins, including the conserved enzyme, ribonuclease III. Ribonuclease III (RNase III) is a double-strand(ds)-specific enzyme that processes diverse RNA precursors in bacterial cells to form the mature, functional forms that participate in protein synthesis and gene regulation. RNase III is involved in the maturation, turnover, and action of small noncoding RNAs (sRNAs), which play key roles in regulating bacterial gene expression in response to environmental inputs and changes in growth conditions. A mass-spectroscopy-based analysis of the E. coli proteome has shown that YmdB and RNase III interact in vivo. However, the functional importance of this interaction is not known. There is preliminary evidence that YmdB regulates RNase III activity during specific stress inputs. Thus, during cellular entry into stationary phase (nutrient limitation), or during the cold shock response, YmdB levels increase, which is correlated with a downregulation of RNase III activity. Inhibition of RNase III may alter the maturation and turnover of sRNAs, as well as other RNAs, during the adaptive response to stress. However, it is unclear whether the inhibition is a direct or indirect effect of YmdB on RNase III activity. Moreover, since YmdB binds ADPR, this (or related) metabolite may influence RNase III activity in an YmdB-dependent manner. If the YmdB-RNase III interaction in fact regulates RNase III, this interaction may connect post-transcriptional regulatory pathways with the cellular metabolic state, as reflected by NAD+ and ADPR levels. The goal of this project is to characterize the YmdB interaction with RNase III, with the long-range goal of understanding the mechanism and role of YmdB regulation of RNase III. Since both YmdB and RNase III are conserved bacterial proteins, characterization of YmdB and its influence on RNase III activity would provide insight on a conserved interaction in bacterial cells in general as well as reveal a potentially novel mechanism of post-transcriptional gene regulation.