A MUTATIONAL-FUNCTIONAL ANALYSIS OF THE ESCHERICHIA COLI MACRODOMAIN PROTEIN, YMDB

Abstract

Gene expression pathways exhibit many “twists and turns,” with theoretically numerous ways in which the pathways can be regulated by both negative and positive feedback mechanisms. A key step in gene expression is RNA maturation (RNA processing), which in the bacterial cell can be accomplished through RNA binding and enzymatic cleavages. The well-characterized bacterial protein Ribonuclease III (RNase III), is a conserved, double-stranded(ds)-specific ribonuclease. In the gram-negative bacterium Escherichia coli, RNase III catalytic activity is subject to both positive and negative regulation. A recent study has indicated that an E. coli protein, YmdB, may negatively regulate RNase III catalytic activity. It has been proposed that YmdB inhibition of RNase III may be part of an adaptive, post-transcriptional physiological response to cellular stress. In E. coli, the model organism in this study, YmdB protein is encoded by the single ymdB gene, and has a predicted molecular mass of ~18.8 kDa. YmdB has been classified as a macrodomain protein, as it exhibits a characteristic fold that specifically provides an ADP-ribose (ADPR) binding site. While YmdB can bind ADPR with good affinity, there may be additional ligands for the binding site. Thus, YmdB protein may interact with other components in the cell, which in turn could modulate the interaction of YmdB with RNase III. In previous research conducted within the Nicholson laboratory at Temple University, affinity-purified Escherchia coli(Ec) YmdB and Aquifex aeolicus (Aa) YmdB were found to exhibit ribonucleolytic activity. This observation initiated the long-term goal of learning how YmdB regulates RNase III, and how the ribonucleolytic activity of YmdB may be involved in this process. The specific goal of this thesis project was to further characterize the ribonucleolytic activity of Ec-YmdB through site-specific mutational analysis. Mutations were introduced into a proposed adenine-binding pocket previously identified by crystallography and by molecular modeling. The adenine-binding pocket is a region within the macrodomain fold where ADP-ribose could bind. The mutations were examined for their effect on Ec-YmdB cleavage of a model RNA, R1.1. The results of this study will contribute to the development of a model describing how the ribonucleolytic activity of YmdB is regulated.