A novel role of ImmE7 in the autoregulatory expression of the ColE7 operon and identification of possible RNase active sites in the crystal structure of dimeric ImmE7

Abstract

Site-specific cleavage of mRNA has been identified in vivo for the polycistronic colicin E7 operon (ColE7), which occurs between G and A nucleotides located at the Asp52 codon (GAT) of the immunity gene (ceiE7). In vitro, this specific cleavage occurs only in the presence of the ceiE7 gene product (ImmE7). The crystal structure of dimeric ImmE7 has been determined at 1.8 A resolution by X-ray crystallographic analysis. We found that several residues located at the interface of dimeric ImmE7 bear surprising resemblance to the active sites of some RNases. These results suggest that dimeric ImmE7 may possess a novel RNase activity that cleaves its own mRNA at a specific site and thus autoregulates translational expression of the downstream celE7 gene as well as degradation of the upstream ceaE7 mRNA.

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

“…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.…”

“…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.…”

“…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.…”

“…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].…”