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Identification and Functional Analysis of Escherichia coli RNase E Mutants  

Shin, Eun-Kyoung (Department of Life Science, Chung-Ang University)
Go, Ha-Young (Department of Life Science, Chung-Ang University)
Kim, Young-Min (Department of Life Science, Chung-Ang University)
Ju, Se-Jin (Department of Life Science, Chung-Ang University)
Lee, Kang-Seok (Department of Life Science, Chung-Ang University)
Publication Information
Korean Journal of Microbiology / v.43, no.4, 2007 , pp. 325-330 More about this Journal
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 and expression of N-terminal domain consisted of 1-498 amino acids (N-Rne) is sufficient to support normal cellular growth. By utilizing these properties of RNase E, we developed a genetic system to screen for amino acid substitutions in the catalytic domain of the protein (N-Rne) that lead to various phenotypes. Using this system, we identified three kinds of mutants. A mutant N-Rne containing amino acid substitution in the S1 domain (I6T) of the protein was not able to support survival of E. coli cells, and another mutant N-Rne with amino acid substitution at the position 488 (R488C) in the small domain enabled N-Rne to have an elevated ribonucleolytic activity, while amino acid substitution in the DNase I domain (N305D) only enabled N-Rne to support survival of E. roli cells when the mutant N-Rne was over-expressed. Analysis of copy number of ColEl-type plasmid revealed that effects of amino acid substitution on the ability of N-Rne to support cellular growth stemmed from their differential effects on the ribonucleolytic activity of N-Rne in the cell. These results imply that the genetic system developed in this study can be used to isolate mutant RNase E with various phenotypes, which would help to unveil a functional role of each subdomain of the protein in the regulation of RNA stability in E. coli.
Keywords
ColE1-type plasmid; degradosome; N-Rne; RNA stability; RNase E;
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