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 efficie ncy of transcription termination at nut (N utilization) sites of vario us bacterial and bacteriophage lambda genes. Similar control mechanism s operate in eukaryotic viruses (e.g. human immunodeficiency virus). A recombinant strain of E. coli producing relatively large amounts of N usB protein (about 10% of cell protein) was constructed. The protein c ould be purified with high yield by anion-exchange chromatography foll owed by gel-permeation chromatography. The protein is a monomer of 15. 6 kDa as shown by analytical ultracentrifugation. Structural studies w ere performed using protein samples labelled with N-15, C-13 and H-2 i n various combinations. Heteronuclear three-dimensional triple-resonan ce NMR experiments combined with a semi-automatic assignment procedure yielded the sequential assignment of the H-1, C-13 and N-15 backbone resonances. Based on experimentally derived scalar couplings, chemical -shift values, amide-exchange data, rind a semiquantitative interpreta tion of NOE data, the secondary structure of NusB has classified as al pha helical, comprising seven alpha helices.