Publication details
NMR study of an RNA polymerase subunit unique for gram-positive bacteria
| Title in English | NMR study of an RNA polymerase subunit unique for gram-positive bacteri |
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| Authors | |
| Year of publication | 2011 |
| Type | Appeared in Conference without Proceedings |
| MU Faculty or unit | |
| Citation | |
| Description | Architecture of RNA polymerase (RNAP) from B. subtilis and other gram-positive bacteria differs from its analogue from gram-negative bacteria in the presence of two additional subunits - delta and omnega1. Recent results indicated that the presence of delta subunit increases the transcription specificity and the efficiency of RNA synthesis. Moreover, the absence of delta subunit is proposed to decrease a virulence of some pathogens. No structural information was available for the delta subunit due to the lack of sequence homology. As crystallization at structure genomic centers failed, we focused on the delta subunit in our NMR structural study. As the previous study showed (López de Saro et al., JMB, 1995), the C-terminal domain of the delta subunit is unstructured and its sequence is highly repetitive. Therefore, we started a systematic investigation of the protein with a shorten construct, corresponding to the well-structured N-terminal part. Its structure was solved using a large set of high-quality NMR restraints, including 2341 NOE (544 long range), 384 RDC, and 33 13C CSA restraints from two aligning media (bacteriophage Pf1, 5% polyacrylamide gel). The calculations were run in CNS using a protocol modified in our lab to combine the SCULPTOR module with RECOORD scripts. Program CING was used to check the quality of the structures. The determined structure allowed us to identify unexpected structure homology of the N-terminal domain of delta subunit with several proteins from the Forkhead DNA/RNA-binding domain SCOP family and to propose residues interacting with the RNAP core. Relaxation dispersion revealed significant slow motions in the interaction surface, supporting the induced-fit model of binding. During last few years many new approaches for study of biologically interesting intrinsically disordered proteins appeared. At the moment we are able to study also the more challenging part of the protein, the C-terminal domain. However, the analysis of the standard spectra was almost impossible due to a very small differences in chemical shifts. Therefore, a new methodology coming from Wiktor Kozminski lab was used to improve the spectra resolution and the full-size protein was then completely assigned. It was a major step for further analysis including secondary structure prediction, study of internal motions or measurement of dipolar couplings. Moreover, the spin labels at different locations are being prepared to describe a behavior of the C-terminal tail even better. The interactions between the delta subunit and the RNAP was studied by NMR titration and gel-shift assay to indicate which subunits are essential for binding of the protein to the core enzyme. |
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