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 all essential Escherichia coli endoribonuclease that plays a major 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 substitution 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 1, 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 1 subdomain, which is spatially distant from the catalytic site posited from crystallographic studies, showed defecting binding to an RNase E substrate, p23 RNA, but still retained RNA cleavage activity-implicating a previously unidentified structural motif in the DNase 1 subdomain in the binding of RNase E to targeted RNA molecules, demonstrating the role of the DNase 1 domain in RNase E activity. subdoinain, which is spatially distant from the Catalytic site posited from ctystallographic studies, showed