NMR STRUCTURAL STUDY OF N-TERMINAL DOMAIN OF RNA POLYMERASE DELTA SUBUNIT FROM Bacillus subtilis

Veronika Motáčková¹, Hana Šanderová², Jiří Nováček¹, Lukáš Žídek¹, Vladimír Sklenář¹, Libor Krásný²

¹National Centre for Biomolecular Research and ²Institute of Experimental Biology, Faculty of Science, Masaryk University, Kotlářská 2, 61137 Brno, Czech Republic

²Institute of Microbiology of the Academy of Sciences of the Czech Republic, v.v.i.,, Vídeňská 1083, 14220 Praha 4-Krč, Czech Republic

Contrary to the well studied RNA polymerases of gram negative bacteria, RNA polymerase of Bacillus subtilis, a gram positive bacterium, contains two additional subunits, referred to as delta and omega1. A well-structured N-terminal domain of subunit delta has been overexpressed in an E. coli expression system, labeled with stable isotopes C-13 and N-15, and investigated by nuclear magnetic resonance.

A standard set of triple resonance NMR experiments was measured and almost all resonances of the protein backbone were assigned. Resonance frequencies of the side-chains were assigned using 3D TOCSY- and NOESY-type spectra. Three-bond coupling constant J(H^NH^α) were obtained from 3D HNHA experiments. Chemical shifts of backbone nuclei, medium range NOEs, and the three-bond coupling constants were analyzed and secondary structure was predicted. Internuclear distance restraints were extracted from NOESY spectra and used in structure calculation.

Acknowledgment:

This work was supported by the Grants MSM0021622413, and LC06030 of the Ministry of Education, Youth, and Physical Culture of the Czech Republic and 204/09/0583 from Czech Science Foundation.