Cloning of the nusA gene of Escherichia coli

Abstract

The EcoRI restriction fragment of wild-type Escherichia coli DNA that can complement the argG mutation was cloned into the EcoRI site on pBR322. The resulting chimeric plasmid (pYN81) contains a 16.0 kilobase fragment and can complement the nusA1 (Friedman 1971) as well as argG mutations. Examination of several deletion derivatives of pYN81 revealed that the activity that complements nusA1 and argG mutations is localized within the 5.3 kilobase segment defined by SalI and BglII sites. When the nusA gene segment was recloned into lambda vector L512, the resulting transducing phages lambda nusA+-1 and lambda nusA+-2 grew normally in nusA1 cells at 40 degrees C or above, unlike the vector phage L512. Proteins encoded by the cloned DNA fragment were examined with minicells containing the chimeric plasmid or UV-irradiated cells infected by the transducing phages. At least six proteins were apparently encoded by the 16.0 kilobase DNA fragment, and genes coding for each of these proteins were localized on the respective restriction segment. One of them with a molecular weight of 64,000 was identified as the nusA gene product. The nusA gene was found to be transcribed counterclockwise with respect to the E. coli genetic map. Endonulease BglII cleaves the gene in the vicinity of the C-terminus, generating a truncated nusA gene product with a molecular weight of 61,000 that can complement the nusA1 mutation.