Solution structure of the RNA polymerase subunit RPB5 from Methanobacterium thermoautotrophicum

Abstract

RPB5 is an essential subunit of eukaryotic and archaeal RNA polymerases. It is a proposed target for transcription activator proteins in eukaryotes, but the mechanism of interaction is not known. We have determined the solution structure of the RPB5 subunit from the thermophilic archeon, Methanobacterium thermoautotrophicum. MtRBP5 contains a four-stranded beta-sheet platform supporting two alpha-helices, one on each side of the beta-sheet, resulting in an overall mushroom shape that does not appear to have any structural homologues in the structural database. The position and conservation of charged surface residues suggests possible modes of interaction with other proteins, as well as a rationale for the thermal stability of this protein.

Figure 1

Alignment of RPB5 from M. thermoautotrophicum (GenBank accession no. O27122) with homologous proteins from the following: Archae: Methanococcus jannaschii (Q58443), Methanococcus vannielii (P41559), Pyrococcus abyssi (CAB49535), Pyrococcus horikoshii (O74019), Thermococcus celer (P31815), Archaeoglobus fulgidus (O28394), Aeropyrum pernix (Q9YAT3), Sulfolobus acidocaldarius (P115210), Thermoplasma acidophilum (Q03588), Halobacterium halobium (P15740). Eukarya: Saccharomyces cerevisiae (NP_009712), Schizosaccharomyces pombe (AF020780), Homo sapiens (CAA11843). Virus: swine fever virus (1097499). Amino acids that are identical and similar in M.t. with at least five other species are highlighted in green and yellow, respectively. The NMR-derived secondary structural elements of mtRPB5 are illustrated above the alignment by using the nomenclature referred to in the text.

Figure 2

NMR-derived structure of mtRPB5. (A) Ribbon diagram of a representative structure. (B) The backbone trace of the 10 lowest energy structures superimposed from residue 12 to 77. Drawn in blue are the solvent-inaccessible sidechains that form the hydrophobic core. The β-strands are cyan, and helices are red. Diagrams were created by using molmol (33).

Figure 3

Strips from the three-dimensional ¹³C-edited NOESY in D₂O at the carbon planes corresponding to the Cγ2 position of Val 25 (19.6 ppm), and the Cδ (131.9 ppm), and Cɛ (116.3 ppm) of Tyr 72 showing unambiguous NOEs between the Tyr 72 and Val 25.

Figure 4

Four views of the surface charge distribution calculated and drawn by using grasp (34), with red representing negative potential (−6.2 kT, full intensity) and blue positive potential (7.6 kT, full intensity). Conserved residues are labeled according to the color scheme in Fig. 1. A is in the same orientation as Fig. 2.