Originally published as Genetics Published Articles Ahead of Print on July 27, 2008.

Genetics, Vol. 179, 1871-1879, August 2008, Copyright © 2008
doi:10.1534/genetics.108.088492

Identification of Amino Acid Residues in the Catalytic Domain of RNase E Essential for Survival of Escherichia coli: Functional Analysis of DNase I Subdomain

Eunkyoung Shin^*,1, Hayoung Go^*,1, Ji-Hyun Yeom^*, Miae Won, Jeehyeon Bae, Seung Hyun Han, Kook Han, Younghoon Lee, Nam-Chul Ha^**, Christopher J. Moore, Björn Sohlberg, Stanley N. Cohen^, and Kangseok Lee^*,2

^* Department of Life Science, Chung-Ang University, Seoul 156-756, Korea, Graduate School of Life Science and Biotechnology, Pochon CHA University, Seongnam 463-836, Korea, Department of Oral Microbiology and Immunology, School of Dentistry, Seoul National University, Seoul 110-749, Korea, Department of Chemistry and Center for Molecular Design and Synthesis, Korea Advanced Institute of Science and Technology, Daejeon 305-701, Korea, ^** National Research Laboratory of Defense Proteins, College of Pharmacy, Pusan National University, Busan 609-735, Korea and Department of Genetics and Department of Medicine, Stanford University, Stanford, California 94305

² Corresponding author: 221 Hueksok-Dong, Donjak-Gu, Department of Life Science, Chung-Ang University, Seoul, Republic of Korea, 156-756.
E-mail: kangseok{at}cau.ac.kr

RNase E is an essential Escherichia coli endoribonuclease that plays a major role in the decay and processing of a large fraction of RNAs in the cell. To better understand the molecular mechanisms of RNase E action, we performed a genetic screen for amino acid substitutions in the catalytic domain of the protein (N-Rne) that knock down the ability of RNase E to support survival of E. coli. Comparative phylogenetic analysis of RNase E homologs shows that wild-type residues at these mutated positions are nearly invariably conserved. Cells conditionally expressing these N-Rne mutants in the absence of wild-type RNase E show a decrease in copy number of plasmids regulated by the RNase E substrate RNA I, and accumulation of 5S ribosomal RNA, M1 RNA, and tRNA^Asn precursors, as has been found in Rne-depleted cells, suggesting that the inability of these mutants to support cellular growth results from loss of ribonucleolytic activity. Purified mutant proteins containing an amino acid substitution in the DNase I subdomain, which is spatially distant from the catalytic site posited from crystallographic studies, showed defective binding to an RNase E substrate, p23 RNA, but still retained RNA cleavage activity—implicating a previously unidentified structural motif in the DNase I subdomain in the binding of RNase E to targeted RNA molecules, demonstrating the role of the DNase I domain in RNase E activity.

International Access Link

Online ISS 1091-6490
Copyright 2008 by the Genetics Society of America
phone: 412-268-1812   fax: 412-268-1813   email: genetics-gsa{at}andrew.cmu.edu