Functional and Structural Studies of Protein Inhibitors of RNase E Activity that Globally Modulate mRNA Abundance in Escherichia coli

Dedication To my parents. Ellington, who allowed me to use his lab for RNase protection assay and growth rate experiment. I acknowledge Drs. sharing strains and antibodies. Many thanks to all the former and current Georgiou group members for their friendship and help. In particular, I thank Meng Zhao for doing the RraA and RraB co-precipitation experiment, Xiaoming Zhan for helping with the RPA and-galactosidase assays, Ji Qiu for helping with the RraA purification, Andrew Hayhurst for helping with the BIACore analysis, Danielle Tullman-Ercek for proofreading part of this dissertation, and Blarcom, and Sylvia Arredondo for sharing the office. Finally I would like to express my deepest appreciation to my best friend and wife Peng Lu for her love, support, help, and thoughtful scientific discussion. Considerable progress has been made during recent years on how the regulation of mRNA abundance affects the level of gene expression. The 118 kDa protein ribonuclease E (RNase E), which was discovered initially by its ability to process E. coli 9S RNA, is now known to have a central role in mRNA decay, the maturation of tRNA, and the degradation of small regulatory RNA. RNase E contains two functionally distinct parts: the N-terminal half (NTH) which is responsible for the catalytic function and the C-terminal half (CTH) which provides a scaffold for the interaction with multiple proteins. Genetic analysis in our lab led to the discovery of RraA and RraB, two transacting protein inhibitors of the E. coli RNase E whose overexpression affects the stability of a large number of transcripts. The work presented here describes the functional and structural characterization of RraA and RraB. The 17.4 kDa protein RraA and the 15.6 kDa protein RraB inhibit the endoribonucleolytic activity of RNase E in vivo and in vitro. Co-precipitation experiments and SPR analysis using BIACore showed that both VII inhibitors physically interact with RNase E with a similar affinity. However, co-precipitation analysis using a set of RNase E mutants containing deletions within the scaffold region of RNase revealed that RraA and RraB bind to different sites on the enzyme. Specifically, RraA may have one or more weak binding sites on the NTH of RNase, but it requires the presence of CTH for high affinity interaction. In contrast, only a short region in the CTH, comprising of residues 694-727, is important for the binding of RraB. Microarray analysis also demonstrated that RraA affects the abundance of …