Studies on the NusB protein of Escherichia coli; Expression and determination of the secondary structure elements by multinuclear NMR spectroscopy

Abstract

The product of the nusB gene of Escherichia coli modulates the efficiency of transcription termination at I Z M ~(N utilization) sites of various bacterial and bacteriophage i 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 "N, "C and *H in various combinations.
Heteronuclear three-dimensional triple-resonance NMR experiments combined with a semi-automatic assignment
procedure yielded the sequential assignment of the 'H, "C and "N 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 (x helical, comprising
seven a helices.