title: Identification of amino acid residues in the catalytic domain of RNase E essential for survival of Escherichia coli: functional analysis of DNase I subdomain.
abstract: 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.
authors.affiliation: Department of Life Science, Chung-Ang University, Seoul, Korea.
authors.forename: Eunkyoung
authors.initials: E
authors.lastname: Shin
authors.forename: Hayoung
authors.initials: H
authors.lastname: Go
authors.forename: Ji-Hyun
authors.initials: JH
authors.lastname: Yeom
authors.forename: Miae
authors.initials: M
authors.lastname: Won
authors.forename: Jeehyeon
authors.initials: J
authors.lastname: Bae
authors.forename: Seung Hyun
authors.initials: SH
authors.lastname: Han
authors.forename: Kook
authors.initials: K
authors.lastname: Han
authors.forename: Younghoon
authors.initials: Y
authors.lastname: Lee
authors.forename: Nam-Chul
authors.initials: NC
authors.lastname: Ha
authors.forename: Christopher J
authors.initials: CJ
authors.lastname: Moore
authors.forename: Björn
authors.initials: B
authors.lastname: Sohlberg
authors.forename: Stanley N
authors.initials: SN
authors.lastname: Cohen
authors.forename: Kangseok
authors.initials: K
authors.lastname: Lee
mesh_terms: D019943:Amino Acid Substitution; D000596:Amino Acids; D020134:Catalytic Domain / Q000235:genetics*; D003850:Deoxyribonuclease I / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004722:Endoribonucleases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D011487:Protein Conformation; D012329:RNA, Bacterial / Q000378:metabolism*; D022661:RNA, Untranslated / Q000378:metabolism
chemical_list: D000596:Amino Acids; D012329:RNA, Bacterial; D022661:RNA, Untranslated; D004722:Endoribonucleases; D003850:Deoxyribonuclease I; C022537:ribonuclease E