IMMUNOAFFINITY PURIFICATION OF THE ESCHERICHIA-COLI RNE GENE-PRODUCT - EVIDENCE THAT THE RNE GENE ENCODES THE PROCESSING ENDORIBONUCLEASE RNASE-E

The rne gene product was highly purified from Escherichia coli cells o verproducing the protein by a procedure including immunoaffinity chrom atography. Expression in vivo and in vitro of the cloned 6-kilobase pa ir DNA fragment containing the entire rne gene resulted in the synthes is of a protein migrating as a 180-kDa polypeptide in the SDS-polyacry lamide gel. The position of the protein on the two-dimensional polyacr ylamide 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 processi ng 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) reveale d that the 180-kDa polypeptide is the only protein recognized by the a ntibodies in a wild type whole cell extract of E. coil. The antibodies cross-reacted with similar molecular weight proteins from a number of different bacteria, suggesting that the rne gene product is evolution arily conserved in the bacterial world.