Escherichia coli 리보핵산 내부분해효소 RNase E의 돌연변이체 선별 및 특성분석

초록

RNase E is an essential Escherichia coli endoribonuclease that plays a major role in the decay and processing ofa large fraction of RNAs in the cell and expression of N-terminal domain consisted of 1-498 amino acids (N-330 Eunkyoung Shin et al. Kor. J. MicrobiolRne) is sufficient to support normal cellular growth. By utilizing these properties of RNase E, we developed agenetic 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 NRne 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. coli cells when the mutant N-Rne was over-expressed. Analysis of copy number of ColE1-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.

키워드