Identification of the dnaQ gene product and location of the structural gene for RNase H of Escherichia coli by cloning of the genes.
By in vitro recombination we have constructed hybrid plasmids capable of complementing a conditional lethal mutator mutation, dnaQ49, in Escherichia coli K12. The dnaQ+ plasmids consist of a full-length pBR322 DNA and a 1.5-kilobase DNA fragment derived from the E. coli chromosome. Specific labeling of plasmid-encoded proteins by the maxicell method revealed that the 1.5-kilobase insert codes for two proteins, one whose molecular weight is 25,000 [the 25-kilodalton (kDal) protein] and the other whose molecular weight is 21,000 (the 21-kDal protein). Because insertion of gamma delta sequence into the dnaQ gene of the plasmid resulted in disappearance of the 25-kDal protein, it was concluded that the 25-kDal protein is the dnaQ gene product. The 21-kDal protein was identified as RNase H on the basis of the following evidence. (i) Cells harboring the dnaQ+ plasmids, with or without the gamma delta insertion in the dnaQ gene, had a 5- to 7-fold higher level of RNase H activity than cells harboring pBR322. (ii) After induction of cells that are lysogenized with dnaQ+-transducing lambda phages, RNase H activity increased considerably. A similar high level of RNase H activity was observed with transducing phages whose dnaQ function was inactivated by insertion of a transposon, Tn3, into the gene, (iii) The plasmid-encoded RNase H, labeled with [35S]methionine, was purified in a manner essentially similar to that of the chromosome-encoded enzyme. These results suggest that the dnaQ gene and the structural gene for RNase H, termed gene rnh, are closely linked and located at 5 min on the linkage map.
D001426:Bacterial Proteins / Q000235:genetics*; D003001:Cloning, Molecular; D004720:Endonucleases / Q000096:biosynthesis / Q000235:genetics*; D004790:Enzyme Induction; D004926:Escherichia coli / Q000235:genetics*; D005796:Genes; D008040:Genetic Linkage; D008970:Molecular Weight; D009154:Mutation; D010957:Plasmids; D016914:Ribonuclease H; D012260:Ribonucleases / Q000096:biosynthesis / Q000235:genetics*
D001426:Bacterial Proteins; D004720:Endonucleases; D012260:Ribonucleases; D016914:Ribonuclease H