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 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.
Taraseviciene
L
L
Department of Microbiology, University of Umeå, Sweden.
Naureckiene
S
S
Uhlin
B E
BE
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
D017931:DNA Primers; D004269:DNA, Bacterial; C042027:RNA I; D012329:RNA, Bacterial; D012313:RNA; D004722:Endoribonucleases; C022537:ribonuclease E