title: Immunoaffinity purification of the Escherichia coli rne gene product. Evidence that the rne gene encodes the processing endoribonuclease RNase E.
abstract: 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.
authors.affiliation: Department of Microbiology, University of Umeå, Sweden.
authors.forename: L
authors.initials: L
authors.lastname: Taraseviciene
authors.forename: S
authors.initials: S
authors.lastname: Naureckiene
authors.forename: B E
authors.initials: BE
authors.lastname: Uhlin
mesh_terms: D001483:Base Sequence; D002846:Chromatography, Affinity; D017931:DNA Primers; D004269:DNA, Bacterial / Q000235:genetics / Q000378:metabolism; D015180:Electrophoresis, Gel, Two-Dimensional; D004591:Electrophoresis, Polyacrylamide Gel; D004722:Endoribonucleases / Q000096:biosynthesis* / Q000302:isolation & purification* / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics*; D015870:Gene Expression; D005798:Genes, Bacterial; D007700:Kinetics; D008961:Models, Structural; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D012313:RNA / Q000737:chemistry / Q000378:metabolism; D012329:RNA, Bacterial / Q000737:chemistry / Q000378:metabolism; D015183:Restriction Mapping; D013379:Substrate Specificity
chemical_list: D017931:DNA Primers; D004269:DNA, Bacterial; C042027:RNA I; D012329:RNA, Bacterial; D012313:RNA; D004722:Endoribonucleases; C022537:ribonuclease E