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Nucleic Acids Research Molecular cloning and nudeotide sequencing of the nusB gene of E . coli

The nusB gene of E.coli has been cloned in plasmid pBR322. Using genetic complementation as an assay for the gene, its location in subclones was analyzed, and the nucleotide sequence of this gene and its flanking regions was determined. The coding region consists of 417 base pairs (bp), which specify a protein of 139 amino acids, and the calculated molecular weight of the nusB protein is 15,702. The nusB protein contains 20 acidic and 21 basic amino acids. A significant promoter sequence was not found to be located in the region upstream from the translational initiation codon. The possibility that the nusB gene consists of an operon is discussed. After the coding region, there is a G-C rich inverted repeat sequence followed by a run of Ts, which could be a transcriptional terminator of the nusB gene. INTRODUCTION The hi gene product of coliphage I regulates phage gene expression by permitting transcription of the phage delayed early and late genes by overcoming transcriptional termination barriers (1). Many E.coli mutants that fail to support the action of f) protein have been isolated, and named nus, an abbreviation for N utilization s_ubstanre (2,3). Recent observations have indicated that the protein products of the nusA and nusB genes are necessary for efficient expression of some bacterial genes as well, including the trp, lac and rpoB-rpoC genes; the proteins are thought to function by modifying the termination of transcription at intragenic transcriptional barriers (4,5;. The nusA gene product, a 69 K dalton protein with an isoelectric point of 4.6, can itself stimulate the synthesis of 8-galactosidase in a coupled in vitro transcription-translation system (6), and can stimulate the "pausing" of RNA polymerase at some transcription termination signals (7-9). The nusB gene product has been reported to be a protein of about 14 K daltons (10,11), but it has not been purified so far. To obtain information on the function of termination and antitennination factors, we cloned and sequenced the nusB gene. © IRL Prets Limited, Oxford, England. 4987 Nucleic Acids Research Figure 1. Structures of pEHl, pEH2 and pEH3 plasmid DNA's. The entire structure of pEHl , pEH2 and pEH3 DNA's are shown. The line indicates the pBR322 vector and the black bar indicates the cloned segment. MATERIALS AND METHODS Bacteria, Bacteriophage, Plasmid and Growth Conditions Strains bearing the nus (K37) and nusB(nusB5) alleles (12) and the specialized transducing bacteriophage XnusB (12), used as a source to clone nusB, were kindly donated by Dr. D.I.Friedman. The pBR322 plasmid was used as a cloning vehicle (13). Cells were grown in L-broth (14). Concentrations of 50ug/ml ampicillin and 20ug/ml tetracycline were used. Enzymes and Chemicals The restriction enzymes, T4 DMA ligase and T4 polynucleotide kinase were purchased from Takara Shuzo Co., Kyoto, Japan. Bacterial alkaline phosphatase was purchased from Boehrmger-Mannheim Co.. (P)Orthophosphate was purchased from New England Nuclear and (Y~ 3 P)ATP was synthesized as described (15). (a-P)UTP (410Ci/mmol) was purchased from Amersham Co.. RNA polymerase was purified by the method of Burgess and Jendrisak (16). Purification of DNA Plasmid DNA was purified essentially by the procedure of Yamamoto e_t a] • (17), by Triton lym^ of cells followed by Sepharose 4B column chromatography. For snail scale preparation of the plasmid, the rapid isolation method of Holmes and Quigley (18) was used. DNA fragments were extracted from agarose gels by the procedure of Yang e_t a_l. (19) or from acrylamide gels by the method of Maxam and Gilbert (20) . Restriction Enzyme Analysis The restriction enzymes used were BamHI, PstI, HindlE, Sail, HaeH , TaqI, Sau3A, Hinfl, Alul and HincH . All these enzymes were used under the conditions specified by the suppliers. Slab gels (2mm thick X 13cm) of polyacrylamide (acrylamide/bisacrylamide, 19 : 1) or la agarose in 90mM Trisborate (pH8.3), 4mM EDTA and 0.5ug/ml ethidium bromide were used for deter-