Identification of amino acid residues in the catalytic domain of RNase E essential for survival of Escherichia coli: functional analysis of DNase I subdomain.
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.
Shin
Eunkyoung
E
Department of Life Science, Chung-Ang University, Seoul, Korea.
Go
Hayoung
H
Yeom
Ji-Hyun
JH
Won
Miae
M
Bae
Jeehyeon
J
Han
Seung Hyun
SH
Han
Kook
K
Lee
Younghoon
Y
Ha
Nam-Chul
NC
Moore
Christopher J
CJ
Sohlberg
Björn
B
Cohen
Stanley N
SN
Lee
Kangseok
K
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
D000596:Amino Acids; D012329:RNA, Bacterial; D022661:RNA, Untranslated; D004722:Endoribonucleases; D003850:Deoxyribonuclease I; C022537:ribonuclease E