Recently, two sequence-specific cleavage sites were found in the ceiE7 gene of the cea-cei-cel polycistronic transcript from the ColE7 operon. The crystal structure of the ColE7 immunity protein (ImE7) suggested that a novel ribonuclease active site is created at the interface of the dimeric structure of the protein. Frame shift mutation of the ceiE7 gene and mutation of histidine residues at the putative active site of the dimeric ImE7 protein respectively abolished and significantly reduced the observed ribonucleolytic cleavage indicating that the dimeric ImE7 protein is indeed involved in this sequence-specific cleavage at the ceiE7 mRNA. It is noteworthy that E. coli S-30 cell extracts must be added to the in vitro reactions in order to detect this ribonucleolytic cleavage. In addition, mutation of the T1 stem-loop structure located between the ceiE7 and the celE7 genes completely turned off the ribonuclease activity in vivo, implying that the T1 stem-loop structure might participate in mediating the formation of a degradosome-like complex required for this specific ribonucleolytic activity.