Nucleic Acids Research, 2005, Vol. 33, No. 19 6439 Figure 3. EMSAs using RNAP subunits E/F from M.jannaschii and human Rpb4/Rpb7. Archaeal E/F and human Rpb4/7 give rise to complexes with the RNA probe in a protein-concentration dependent manner. Asterisks indicate the position of the free probe, while double asterisks indicate the position of the protein–RNA complexes. The protein concentrations were 25 ng, 50 ng, 100 ng, 250 ng, 500 ng, 1 mg, 2 mg, 3.5 mg, 5 mg, 7.5 mg and 10 mg. (A) Lane 1, free probe; lanes 2–12: increasing concentration of M.jannaschii E/F complex. (B) Quantification of the EMSA experiment for the M.jannaschii complex has been carried out by plotting the fractional occupancy of the probe bound to the protein, corrected for the background, against the protein concentration (C) Lane 1, free probe; lanes 2–10: increasing concentration of human Rpb4/Rpb7 complex. (D) Quantification of the EMSA experiment for the human complex has been carried out as described in (B). using increasing protein concentrations (25 ng to 10 mg) and the mixture analysed by PAGE (Figure 3A) to monitor the formation of protein–RNA complexes. A quantitative analysis of the RNA binding gave a value of 540 nM for the dissoci- 5 ation constant (Figure 3B). A similar procedure was carried out with the recombinant human Rpb4/Rpb7 heterodimer and gave an RNA dissociation constant of 420 nM (Figure 3C and D). Protein–RNA complexes with comparable afﬁnities were obtained using various RNA probes (data not shown) 10 indicating that the binding is not sequence speciﬁc. Dissecting the RNA-binding region in the archaeal E/F complex To identify the RNA-binding surface of the M.jannaschii E/F complex we mutated residues of the E subunit, tested the 15 RNA-binding properties of the mutant protein by a gel-shift assay and compared the binding with the wild-type protein. Targets for the mutagenesis were selected based on their surface-exposure (in the context of RNAPII), amino acid con- servation (in the archaeal domain) and chemical nature. Because we expected a reasonably large interaction area 20 between protein and nucleic acid, we reasoned that substitu- tion of one of the contact residues to alanine was probable to have a minor effect on RNA binding owing to the large num- ber of remaining interactions stabilizing the complex. We therefore opted for charge-reversal substitutions which are 25 more probable to disrupt the interaction. The atomic model of the E subunit shows the presence of two distinct domains forming an elongated structure (Figure 2). The sequence of the C-terminal domain (Figure 2, upper blue domain) indicated the presence of a S1 motif (Figure 1, strands 30 B1–B6). The S1 motif folds into the b-barrel structure which characterizes OB folds. A cluster of conserved surface- exposed amino acids is located on the exposed face of the b-barrel, and includes charged or polar residues (Asp105, His109, Ser111, Gln112, Lys152 and Lys160) and aromatic 35 residues (Phe95 and Phe98) (Figure 4). To determine whether any of these surface residues were functionally important, each of them was replaced by either a reverse-charge mutation or by alanine. In some OB folds, the loop connecting strand B4 and B5 has been shown to play a central role in DNA or RNA 40