Nucleic Acids Research, 2005, Vol. 33, No. 19 6441 Figure 5. Results of EMSAs performed using a radiolabelled RNA probe. (A) Example of a mobility assay using the M.jannaschii E/F heterodimer. Lane 1, free probe; lanes 2–8, increasing concentrations of the E/F complex with a F95A mutation in the E subunit; lanes 10–15, increasing concentrations of the E/F complex with a D105K mutation in the E subunit. (B) Example of a mobility assay using the M.jannaschii E/F heterodimer. Lane 1, free probe; lanes 2-8, increasing concentrations of the E/F complex with a Q112K mutation in the E subunit; lanes 10–15, increasing concentrations of the E/F complex with the Dloop mutation in the E subunit. (C) Summary of the effect of mutations in the M.jannaschii E subunit on the RNA-binding properties. For each mutant protein, the percentage of RNA probe bound to the protein, after background correction, is shown. (D) Example of a mobility assay performed using the human Rpb4/Rpb7 heterodimer. Lane 1, free probe; lanes 2–7, increasing concentrations of wild-type Rpb4/Rpb7 (ranging from 125 ng to 10 mg); lanes 9–14, increasing concentrations of Rpb4/Rpb7 with a H14E mutation in Rpb7. (E) Example of a mobility assay performed using the human Rpb4/Rpb7 heterodimer. Lane 1, free probe; lanes 2-8, increasing concentrations of Rpb4/Rpb7 with a D153K mutation in Rpb7; lanes 10–15, increasing concentrations of Rpb4/Rpb7 with a F158A mutation in Rpb7. (F) Summary of the effect of mutations in the human Rpb7 subunit on the RNA-binding properties, where experimental data were treated as described for the M.jannaschii E subunit. the human structure (residues 21 and 22 in yeast, Figure 1) are well conserved and residues 14–28 at the N-terminus of the human Rpb4 overlap with the corresponding residues (21–35) of the yeast structure. The conformation of this loop is 5 probable to be conserved in eukaryotes as it is stabilized by the interactions of conserved hydrophobic side chains: Phe23 and Phe26 of Rpb4 pack against Phe95 of Rpb4 and Phe80 of Rpb7. In the crystal structure of the human protein, the loop is directly followed by strand A10 in which the poly- 10 peptide chain forms hydrogen bonds with strand A1 of the Rpb7 subunit. The yeast Rpb4 sequence has a 11-residue insertion at this position (residues 36–46) that folds to form a pair of short antiparallel b-strands before entering strand A10. In the structure of the 12-subunit yeast polymerase, the loop 15 between strands A3 and A4 of Rpb7 (the ‘tip loop’) contacts the core subunits of the polymerase. This loop region appears to be ﬂexible as it is disordered in the high-resolution yeast crystal structure (16) and adopts slightly different conforma- tions in the different copies of the Rpb7 subunit within the 20 crystallographic asymmetric units of both the archaeal and human structures. The S1 motif, present in the C-terminal domain of the E subunits, is found in a number of other proteins including bacterial and eukaryotic translation initiation factors, proteins 25 involved in mRNA degradation, proteins involved in mRNA release from the spliceosome as well as bacterial transcription factors. Some of these proteins have been shown to bind