| Description |
RNA polymerase (RNAP) from gram-positive bacteria such as Bacillus subtilis differs from well-studied RNA polymerase from gram-negative bacteria in the presence of two additional subunits, delta and omega1. Recent results indicated that the presence of delta subunit increases the transcription specificity and the efficiency of RNA synthesis. On the other hand, the absence of delta subunit is proposed to decrease a virulence of some pathogens. As crystallization at structure genomics centers failed, we focused on NMR studies of the delta subunit to describe its structure and internal dynamics. As the previous study showed (López de Saro et al., JMB, 1995), the C-terminal domain of the delta subunit is unstructured and its repetitive sequence is highly acidic. Therefore, we started a systematic investigation of the protein with a shorten construct, corresponding to the well-structured N-terminal part and published its high-quality structure. The more challenging part of the protein, the C-terminal domain, was not initially studied because the NMR methodology for disordered, flexible proteins was not sufficiently developed at the beginning of the project. Fortunately, many new approaches for study of biologically interesting intrinsically disordered proteins appeared during the last few years. In contrast to X-ray crystallography, NMR can provide valuable information on residual secondary structure, possible transient contacts beyond the limit of the nuclear Overhauser, and internal dynamics of the disordered polypeptide chain. The full-length delta protein was prepared using a standard protocol of overexpression in the E.coli system to produce a 15N,13C-uniformly labeled sample. The basic spectra, including a standard set of triple resonance NMR experiments, 3D TOCSY, and 3D NOESY, were measured on a 600MHz spectrometer. However, the analysis of the spectra was almost impossible due to a very small differences in chemical shifts. Therefore, a new methodology was implemented to improve the spectra resolution and the full-size protein was then completely assigned. The obtained assignment allowed us to predict secondary structure propensities of the disordered domain, probe its conformational behavior using paramagnetic labels, and study internal motions of the whole construct. Moreover, we recalculated the 3D structure of the full-length protein to confirm a relevance of the structural model of its N-terminal domain determined within the frame of the initial study of delta protein
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