Studies on the NusB protein of Escherichia coli--expression and determination of secondary-structure elements by multinuclear NMR spectroscopy.
The product of the nusB gene of Escherichia coli modulates the efficiency of transcription termination at nut (N utilization) sites of various bacterial and bacteriophage lambda genes. Similar control mechanisms operate in eukaryotic viruses (e.g. human immunodeficiency virus). A recombinant strain of E. coli producing relatively large amounts of NusB protein (about 10% of cell protein) was constructed. The protein could be purified with high yield by anion-exchange chromatography followed by gel-permeation chromatography. The protein is a monomer of 15.6 kDa as shown by analytical ultracentrifugation. Structural studies were performed using protein samples labelled with 15N, 13C and 2H in various combinations. Heteronuclear three-dimensional triple-resonance NMR experiments combined with a semi-automatic assignment procedure yielded the sequential assignment of the 1H, 13C and 15N backbone resonances. Based on experimentally derived scalar couplings, chemical-shift values, amide-exchange data, and a semiquantitative interpretation of NOE data, the secondary structure of NusB has classified as alpha helical, comprising seven alpha helices.
Berglechner
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F
Lehrstuhl für Organische Chemie und Biochemie, Technische Universität München, Garching, Germany.
Richter
G
G
Fischer
M
M
Bacher
A
A
Gschwind
R M
RM
Huenges
M
M
Gemmecker
G
G
Kessler
H
H
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D001483:Base Sequence; D002455:Cell Division; D002852:Chromatography, Ion Exchange / Q000379:methods; D004926:Escherichia coli / Q000235:genetics*; D029968:Escherichia coli Proteins; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics; D014157:Transcription Factors / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism
D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; C074952:NusB protein, E coli; D011994:Recombinant Proteins; D014157:Transcription Factors