Immunoaffinity purification of the Escherichia coli rne gene product: Evidence that the rne gene encodes the processing endoribonuclease RNase E super

The rne gene product was highly purified from Escherichia coli cells overproducing the protein by a procedure including immunoaffinity chromatography. Expression in vivo and in vitro of the cloned 6-kilobase pair DNA fragment containing the entire rne gene resulted in the synthesis of a protein migrating as a 180-kDa polypeptide in the SDS-polyacrylamide gel. The position of the protein on the two-dimensional polyacrylamide gel indicated that the protein is highly acidic. The enzymatic activity test which used as the substrate RNA I and 9 S RNA provided evidence that the rne gene is the structural gene for the RNA processing enzyme RNAse E. The Western blot analysis performed using a rabbit antiserum raised against a truncated 110-kDa protein fragment of RNase E (containing two-thirds of the sequence from the N terminus) revealed that the 180-kDa polypeptide is the only protein recognized by the antibodies in a wild type whole cell extract of E. coli. The antibodies cross-reacted with similar molecular weight proteins from a number of different bacteria, suggesting that the rne gene product is evolutionarily conserved in the bacterial world.