Summary
The nucleotide sequence of the entire nusB gene of Escherichia coli has recently been determined and the amino acid sequence of its product deduced (Ishii et al. 1984; Swindle et al. 1984). The NusB protein was purified by chromatography on Sephadex G-100, phosphocellulose and hydroxylapatite. Purification of the protein was monitored using 14C-labelled NusB protein, which was synthesized in a maxicell containing an nusB plasmid as a marker. The final product, which was at least 95% pure as judged by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate, had a molecular weight of about 16,000 and an isoelectric point of about 7.3. Analytical data on the amino acid composition of the purified protein agreed with that deduced from the DNA sequence and indicated that this protein was indeed the product of the nusB gene.
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Abbreviations
- SDS:
-
sodium dodecyl sulphate
- kDa:
-
kilodaltons
- bp:
-
base pair(s)
- kbp:
-
kilobase pair(s)
References
Bittner M, Vapnek D (1981) Versatile cloning vectors derived from the runaway-replication plasmid pKN402. Gene 15:319–329
Blombäck B, Blomback M, Edman P, Hessel B (1966) Human fibronopeptides isolation, characterization and structure. Biochim Biophys Acta 115:371–396
Bonner WM, Laskey RA (1974) A film detection method for tritium-labelled proteins and nucleic acids in polyacrylamide gels. Eur J Biochem 46:83–88
Farnham PJ, Greenblatt J, Platt T (1982) Effects of musA protein on transcription termination in the tryptophan operon of Escherichia coli. Cell 29:945–951
Friedman DI, Jolly CT, Mural RJ (1973) Interference with the expression of the N gene product of phage λ in a mutant of Escherichia coli. Virology 51:216–226
Friedman DI, Bauman M, Baron LS (1976) Cooperative effects of bacterial mutations affecting λ N gene expression: 1. Isolation and characterization of a nusB mutant. Virology 73:119–127
Greenblatt J, Li J (1981a) The nusA gene protein of Escherichia coli: Its identification and a demonstration that it interacts with the gene N transcription anti-termination protein of bacteriophage lambda. J Mol Biol 147:11–23
Greenblatt J, Li J (1981b) Interaction of the sigma factor and the nusA gene protein of Escherichia coli with RNA polymerase in the initiation-termination cycle of transcription. Cell 24:241–248
Greenblatt J, Li J, Adhya S, Friedman DI, Baron LS, Redfield B, Kung HF, Weissbach H (1980a) L factor that is required for β-galactosidase synthesis is the nusA gene product involved in transcription termination. Proc Natl Acad Sci USA 77:1991–1994
Greenblatt J, Malnoe P, Li J (1980b) Purification of the gene N transcription anti-termination protein of bacteriophage λ. J Biol Chem 255:1465–1470
Greenblatt J, McLimont M, Hanly S (1981) Termination of transcription by nusA gene protein of Escherichia coli. Nature 292:215–220
Hase T, Matsubara H, Hutber GN, Rogers LJ (1982) Amino acid sequence of Nostoc strain MAC ferredoxin I and II. J Biochem 92:1347–1355
Heinemann SF, Spiegelman WG (1970) Role of the gene N product in phage lambda. Cold Spring Harbor Symp Quant Biol 35:315–318
Imamoto F, Ishii S, Maekawa T, Kuroki K, Kano Y, Miyashita T (1983) Effect of nusA protein on in vitro transcription of the tryptophan operon. In: Schlessinger D (ed) Microbiology. American Society of Microbiology, Washington DC, p 57
Ishii S, Hatada E, Maekawa T, Imamoto F (1984) Molecular cloning and nucleotide sequencing of the nusB gene of E. coli. Nucleic Acids Res 12:4987–4995
Ishii S, Kuroki K, Sugino Y, Imamoto F (1980) Purification and characterization of the N gene product of bacteriophage lambda. Gene 10:291–300
Kassavetis G, Chamberlin M (1981) Pausing and termination of transcription within the early region of bacteriophage T7 DNA in vitro. J Biol Chem 256:2777–2786
Kumar S, Bovre K, Guha A, Hradecna Z, Mahr SVM, Szybalski W (1969) Orientation and control of transcription in E. coli phage λ. Nature 221:823–825
Kung HF, Spears C, Weissbach H (1975) Purification and properties of a soluble factor required for the deoxyribonucleic acid-directed in vitro synthesis of β-galactosidase. J Biol Chem 250:1556–1562
Kuroki K, Ishii S, Kano Y, Miyashita T, Nishi K, Imamoto F (1982) Involvement of the nusA and nusB gene products in transcription of Escherichia coli tryptophan operon in vitro. Mol Gen Genet 185:369–371
Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 277:680–685
Lozeron HA, Dahlberg JE, Szybalski W (1976) Processing of the major leftward mRNA of coliphage lambda. Virology 71:262–277
Luzzati D (1970) Regulation of λ exonuclease synthesis: Role of the N gene product and λ repressor. J Mol Biol 49:515–519
O'Farrell PH (1975) High resolution two-dimensional electrophoresis of transcription in the N operon of bacteriophage lambda. Mol Gen Genet 117:72–81
Roberts JW (1969) Termination factor for RNA synthesis. Nature 224:1168–1174
Rosenberg M, Court D, Shimatake H, Brady C, Wulff DL (1978) The relationship between function and DNA sequence in an intercistronic regulatory region of phage λ. Nature 272:414–423
Sancar A, Hack AM, Rupp WD (1979) Simple method for identification of plasmid-coded proteins. J Bacteriol 137:692–693
Sheehan JC, Yang DDH (1958) The use of N-formylamino acids in peptide synthesis. J Am Chem Soc 80:1154–1158
Skalka A, Butler B, Echols H (1967) Genetic control of transcription during development of phage λ. Proc Natl Acad Sci USA 58:576–583
Starauch M, Friedman DI (1981) Identification of the nuB gene product of Escherichia coli. Mol Gen Genet 182:498–501
Sugino Y, Tsumasawa S, Yutani K, Ogasawara K, Suzuki M (1980) Amino-terminally formylated tryptophan synthetase α-subunit produced by the trp operon cloned in a plasmid vector. J Biochem 87:351–354
Swindle J, Ajioka J, Georgopoulos C (1981) Identification of the E. coli groNB(nusB) gene product. Mol Gen Genet 182:409–413
Swindle J, Ajioka J, Dawson D, Myers R, Carrol D, Georgopoulos C (1984) The nucleotide sequence of the Escherichia coli K12 nusB (groNB) gene. Nucleic Acids Res 12:4977–4985
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Communicated by M. Takanami
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Maekawa, T., Nagase, T., Imamoto, F. et al. Purification of the NusB gene product of Escherichia coli K12. Molec. Gen. Genet. 200, 14–20 (1985). https://doi.org/10.1007/BF00383306
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DOI: https://doi.org/10.1007/BF00383306