STRUCTURE AND FUNCTION OF THE DNA-DEPENDENT RNA-POLYMERASE OF SULFOLOBUS

The DNA-dependent RNA polymerase of the thermophilic archaeon Sulfolob us acidocaldarius (RNAP, EC 2.7.7.6) was shown to consist of thirteen components ranging in size from 122 kDa to approximately 5 kDa. They w ere named B, A', A'', D, E, F, G, H, I, K, L, M and N. The staining in tensities indicate that they occur in an equimolar ratio. The complexi ty of the enzyme resembles that of eucaryal RNA polymerases. Most RNAP components and two subassemblies containing the components D plus L a nd E plus I could be separated in electrophoresis on a cellulose aceta te support. The D-L subunit-complex exhibited a typical yellow colour with an absorbance maximum of 400 nm. Inductive coupled plasma mass sp ectroscopy showed the absence of heavy metal atoms in this complex, bu t the presence of two zinc ions per RNAP molecule. The subunit G is ph osphorylated. The component F was separated into five subspecies diffe ring in charge density. By anion exchange chromatography an F-free RNA P and five variants, each containing a different F-component (Fg to Fl ) were separated. Renaturation of total dissociated RNAP led to an act ivity recovery of up to 30%. On this basis experiments to reconstitute a functional enzyme from the separated subunits were done. The compon ents A'', E, H and It appeared to be required for the basic activity o f the RNAP. Crystallization of the RNAP yielded needle shaped and rhom boid crystals of a size of up to 0.5 mm. Only F-containing RNAP could be crystallized, but no x-ray diffraction was obtained so far. Genes e ncoding small subunits of the RNAP of S. acidocaldarius were compared to genes of small components of the eucaryal polymerases A (I), B (II) and C (III). Subunit It was shown to be homologous to the eucaryal co mponent ABC23, N to ABC10 beta and L to AC19. No homologies to bacteri al subunits were found. The components E, F and G have so far no bacte rial or eucaryal counterparts. In contrast to the genes of the subunit s H, B, A' and A'', which are transcribed jointly, the genes encoding the smaller subunits were transcribed separately. A further hint to a close relationship between archaeal and eucaryal transcription machine ry was the discovery of a gene directly downstream of subunit L, which had a high similarity to the eucaryal transcription factor TFIIS. In vitro transcription of the 16S/23S rRNA-promoter of S. shibatae, the p romoter of transcript 3 of the Sulfolobus virus SSV1 and of mutant der ivatives with a cell-free extract or with purified RNAP of S. shibatae showed that the RNAP is able to recognize the correct start site by i tself and independent of Box A, whereas with a cell-free extract the s tart point of transcription was determined by the position of box A. W e conclude that one or more factors present in the cell-free extract a re involved in box A directed transcription-initiation.