Characterization of the specific cleavage of ceiE7-mRNA of the bactericidal ColE7 operon

Abstract

Posttranscriptional control of the bactericidal ColE7 operon has been implicated by a feedback endonucleolytic cleavage of its own mRNA. The cleavage site has been located at the coding region of ceiE7, the second cistron of the ColE7 cea-cei-cel polycistronic transcript. Interestingly, Im7 protein, the translation product of ceiE7, is required for the specific cleavage. It was found that both sequence (GAUCUGAUU) flanking the cleavage site and the putative T1 stem-loop structure distal to the coding region of ceiE7 gene play a critical role for the specific cleavage of ceiE7-mRNA. Furthermore, we have verified that a di-nucleotide GG sequence located at the topmost position of the loop region of the putative stem-loop structure is essential for the specific cleavage of ceiE7-mRNA. Thus, our data reveal the existence of a novel mRNA degradative machinery for the regulation of the expression of ColE7 operon. Ó 2002 Elsevier Science (USA). All rights reserved.Keywords: Colicin E7; mRNA decay; Specific cleavage of mRNA; Stem-loop structure of RNA The ColE operon comprises the colicin structural (cea), immunity (cei), and lysis genes (cel). Bactericidal activities of colicins have been classified as DNase (colicin E2, E7, E8, and E9), RNase (colicin E3, E6, and cloacin DF13), ionophore (colicin E1, A, N, K, Ia, and B), inhibition of protein synthesis (colicin E5 and D), and inhibition of synthesis of murein and LPS (colicin M) [1,2]. Immunity protein (Im) encoded by the cei gene is hostÕs antidote for colicin [3][4][5]. Lys protein (or bacteriocin-release protein, BRP), the cel gene product, activates phospholipase A in the outer membrane of Escherichia coli cells and consequently alters the permeability of the outer membrane and facilitates colicin release from the periplasm [6]. Overexpression of cea and cel is lethal to the host; therefore, a tightly controlled SOS promoter is utilized to safeguard the host cells containing the ColE operon [7].We recently reported that immunity protein (Im7) of colicin E7 mediates a site-specific cleavage of its own mRNA [8]. The crystal structures of monomeric [9] and dimeric forms [8] of Im7 protein have been resolved. Most interestingly, it was found that the monomeric form of Im7 is a DNase inhibitor, while the dimeric form may perform a putative novel RNase activity. Thus a novel autoregulatory expression of the cea-cei-cel polycistronic transcript of the ColE7 operon has been proposed [8].In prokaryotes, the rate-limiting step in mRNA decay is usually an initial endonucleolytic cleavage, followed by rapid 3 0 -5 0 exoribonuclease degradation [10][11][12]. Two endoribonucleases, RNase III and RNase E [13,14], are largely responsible for bulk mRNA processing at either the 5 0 untranslated region (UTR) [15][16][17] or 3 0 UTR [18], while RNase II and polynucleotide phosphorylase [19] are mainly responsible for bulk mRNA degradation in a fashion of 3 0 -5 0 direction into mono-nucleotides. It has been shown that the consensus sequence at the cleavage site of RNase E is ...

A Sequence-Specific RNase Activity Derived from the Interface of the Dimeric Immunity Protein of the ColE7 Operon

“…A concomitant minor specific cleavage occurs between nucleotides A and U of codon Ile54 (Fig. 1B) [10]. Moreover, our site-directed mutagenesis experiment revealed that the sequence comprised between the two cleavage sites (G AUCUGA UU) is essential for the specificity of the cleavage [10].…”

“…1B, our unpublished data). Substitution of the GG dinucleotide at the top position of the loop by CC completely abolishes the occurrence of the specific cleavage at the coding region of ceiE7 mRNA [10] and results in substantial reduction of translation of the celE7 mRNA. This result clearly indicated that the T1 stem-loop structure is essential for the specific cleavage of the ceiE7 mRNA.…”

“…Furthermore, we discovered in a previous study that two specific cleavages *Address correspondence to this author at the Institute of Biochemistry, National Yang Ming University, No. 155, section 2, Li-Nong street, PeiTou, Taipei 11221, Taiwan; Tel: +886-2-28267129; Fax: +886-2-28264843; E-mail: kfchak@ym.edu.tw occur within the ceiE7 mRNA of the polycistronic transcript [9,10] and showed that the sequence (AUCUGA) situated between these two cleavage sites plays a critical role for the specificity of the cleavage (Fig. 1B).…”

Bet-hedging in bacteriocin producing Escherichia coli populations: the single cell perspective

“…We predicted that these expressers persist until enough lysis proteins accumulate to lyse the producer cell, releasing colicins into the media. However, we hypothesised that different colicinogenic strains would diverge in their expression patterns in accordance with the differences in their regulatory elements 16 26 27 28 and modes of action 6 .…”