Structural study of the N-terminal domain of the alpha subunit of Escherichia coli RNA polymerase solubilized with non-denaturing detergents

The amino-terminal domain of the alpha subunit (alpha NTD) of Escherichia c oli RNA polymerase consisting of 235 amino acid residues functions in the a ssembly of the alpha, beta, and beta' subunits into the core-enzyme. It has a tendency to form aggregates by itself at higher concentrations. For NMR structural analysis of alpha NTD, the solution conditions, including the us e of non-denaturing detergents, were optimized by monitoring the translatio nal diffusion coefficients using the held gradient NMR technique. Under the optimal conditions with taurodeoxycholate and with the aid of deuteration of the sample, alpha NTD gave triple-resonance spectra of good quality, whi ch allowed the assignment of a large part of the backbone resonances. Analy sis of the pattern of NOEs observed between the backbone amide and alpha-pr otons demonstrated that alpha NTD has three alpha-helices and two beta-shee ts. Although the secondary structure elements essentially coincide with tho se in the crystal structure, the larger of the two beta-sheets has two addi tional beta-strands. The irregular NOE patterns observed for the three posi tions in the beta-sheets suggest the presence of beta-bulge structures. The positions of the three helices coincide with the conserved sequence region s that are responsible for the subunit assembly.