2000 Volume 128 Issue 2 Pages 337-344
The amino-terminal domain of the α subunit (αNTD) of Escherichia coli RNA polymerase consisting of 235 amino acid residues functions in the assembly of the α, β, and β' subunits into the core-enzyme. It has a tendency to form aggregates by itself at higher concentrations. For NMR structural analysis of αNTD, the solution conditions, including the use of non-denaturing detergents, were optimized by monitoring the translational diffusion coefficients using the field gradient NMR technique. Under the optimal conditions with taurodeoxycholate and with the aid of deuteration of the sample, αNTD gave triple-resonance spectra of good quality, which allowed the assignment of a large part of the backbone resonances. Analysis of the pattern of NOEs observed between the backbone amide and α-protons demonstrated that αNTD has three α-helices and two β-sheets. Although the secondary structure elements essentially coincide with those in the crystal structure, the larger of the two β-sheets has two additional β-strands. The irregular NOE patterns observed for the three positions in the β-sheets suggest the presence of β-bulge structures. The positions of the three helices coincide with the conserved sequence regions that are responsible for the subunit assembly.
