Protein-protein interactions in the archaeal transcriptional machinery: binding studies of isolated RNA polymerase subunits and transcription factors.
Transcription in Archaea is directed by a pol II-like RNA polymerase and homologues of TBP and TFIIB (TFB) but the crystal structure of the archaeal enzyme and the subunits involved in recruitment of RNA polymerase to the promoter-TBP-TFB-complex are unknown. We described here the cloning expression and purification of 11 bacterially expressed subunits of the Pyrococcus furiosus RNAP. Protein interactions of subunits with each other and of archaeal transcription factors TFB and TFB with RNAP subunits were studied by Far-Western blotting and reconstitution of subcomplexes from single subunits in solution. In silico comparison of a consensus sequence of archaeal RNAP subunits with the sequence of yeast pol II subunits revealed a high degree of conservation of domains of the enzymes forming the cleft and catalytic center of the enzyme. Interaction studies with the large subunits were complicated by the low solubility of isolated subunits B, A', and A'', but an interaction network of the smaller subunits of the enzyme was established. Far-Western analyses identified subunit D as structurally important key polypeptide of RNAP involved in interactions with subunits B, L, N, and P and revealed also a strong interaction of subunits E' and F. Stable complexes consisting of subunits E' and F, of D and L and a BDLNP-subcomplex were reconstituted and purified. Gel shift analyses revealed an association of the BDLNP subcomplex with promoter-bound TBP-TFB. These results suggest a major role of subunit B (Rpb2) in RNAP recruitment to the TBP-TFB promoter complex.
Goede
Bernd
B
Lehrstuhl für Allgemeine Mikrobiologie, Universität Kiel, am Botanischen Garten 1-9, 24107 Kiel, Germany.
Naji
Souad
S
von Kampen
Oliver
O
Ilg
Karin
K
Thomm
Michael
M
D019843:Archaeal Proteins / Q000502:physiology; D020134:Catalytic Domain; D012321:DNA-Directed RNA Polymerases / Q000737:chemistry* / Q000378:metabolism; D008957:Models, Genetic; D008958:Models, Molecular; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D025941:Protein Interaction Mapping / Q000379:methods*; D017434:Protein Structure, Tertiary; D020027:Pyrococcus furiosus / Q000201:enzymology*; D012319:RNA Polymerase II / Q000737:chemistry; D014157:Transcription Factors / Q000737:chemistry*; D014158:Transcription, Genetic
D019843:Archaeal Proteins; D014157:Transcription Factors; D012319:RNA Polymerase II; D012321:DNA-Directed RNA Polymerases