6438 Nucleic Acids Research, 2005, Vol. 33, No. 19 Figure 2. Structures of the M.jannaschii E/F complex (PDB entry 1go3), yeast Rpb4/Rpb7 complex (PDB entry 1wcm) and human Rpb4/Rpb7 complex. The complexes are viewed from the side of the heterodimer which faces the RNA exit groove of the RNAPII core. The structurally conserved regions of the Rpb4 subunits are coloured pink while the structurally conserved regions of the Rpb7 subunits are coloured violet. In the M.jannaschii crystal structure no electron density is visible for the loop connecting strands B4 and B5; this loop has been drawn for the sake of clarity and is coloured in dark blue. Regions of the yeast and archaeal structures that differ from the human protein are shown in grey. The yeast structure is from the 12-subunit RNAPII complex obtained at 3.8 s (PDB entry 1wcm) rather than the higher resolution but less complete structure of the isolated heterodimer. This figure and all others representing molecular structures were generated with PyMOL (http://pymol.sourceforge.net). RNA binding The probe template was a gift from S. Curry (Imperial College) and directs transcription in pGEM4Z (Promega) of a 54-ribonucleotide RNA species. The RNA probe was pro- 5 duced by in vitro transcription using T7 RNA polymerase and 32P-UTP according to standard procedures. Following tran- scription, the template was digested with 1 U of RNase-free DNase I and unincorporated nucleotides removed using a MicroSpin G-25 desalting column (Amersham Biosciences); 10 the RNA was recovered by precipitation with 1 vol of 7.5 M NH4OAc and 4 vol of ethanol. Probe at a concentration of 2500 c.p.m. was used in a 10 ml reaction containing 10 mM MOPS, pH 6.8, 100 mM NaCl, 10% glycerol, 40 U of RNasin (Promega) and varying amounts of protein. The samples were 15 incubated at room temperature for 15 min before loading onto a native 4–20% acrylamide (Novex), 0.5· TBE gel. The com- plexes were separated electrophoretically on 4–20% TBE gels (Invitrogen) at 100 V for 2.5 h at 4 C and quantiﬁed using a phosphorimager (Fuji). 20 RESULTS Crystal structure of the human Rpb4/Rpb7 heterodimer The human Rpb4/Rpb7 complex crystallizes with four copies of the heterodimer in the crystallographic asymmetric unit. The structure was solved at 2.7 s resolution by molecular 25 replacement using the yeast Rpb4/Rpb7 complex as a search model (16) and reﬁned to a free R-factor of 26.6% (working R-factor of 23.1%) with good geometry (for more statistics see Table 1). The Rpb7 polypeptide folds into two separate domains forming an elongated structure (Figure 2, blue). The six helices 30 of Rpb4 (Figure 2, pink) pack around the central region of Rpb7 at the interface between the two domains. The N-terminal domain of Rpb7 (lower blue domain in Figure 2) folds into a four-stranded anti-parallel b-sheet (strands A1– A4) that wraps around an a-helix (K2). An additional ﬁfth 35 strand of this b-sheet (strand A10) is formed from residues near the N-terminus of Rpb4. The C-terminal domain of Rpb7 folds into a b-barrel structure known as an OB fold (strands B1–B6) which is capped by a small 3-stranded antiparallel b-sheet (strands C1–C3). 40 As expected, the structure of the human Rpb4/Rpb7 het- erodimer is similar to the yeast and M.jannaschii orthologues (Figure 2). Optimally superimposition of the human Rpb4/ Rpb7 heterodimer onto the yeast heterodimer in the 12-subunit RNAPII complex obtained at 3.8 s (PDB entry 45 1wcm) results in 282 equivalent Ca atoms that are within 4 s (r.m.s. separation of 0.66 s). A similar superposition onto the higher resolution (2.3 s) but less complete crystal structure of the isolated yeast heterodimer (PDB entry 1y14) results in 249 Ca atoms within 4 s (rmsd 0.61 s). Superposi- 50 tion onto the archaeal heterodimer (PDB entry 1go3) results in 252 equivalent Ca atoms that are within 4 s (rmsd of 0.99 s). The archaeal E/F and the human Rpb4/Rpb7 heterodimers bind RNA with comparable affinities To test for ssRNA-binding activity, we performed electro- 55 phoretic mobility shift assays (EMSA). The archaeal E/F heterodimer was incubated with a radiolabelled RNA probe