6440 Nucleic Acids Research, 2005, Vol. 33, No. 19 Figure 4. Ribbon diagrams showing the position of the residues mutated in this study on the crystal structures of M.jannaschii subunit E and human Rpb7. The subunits are viewed in the same orientation as that used in Figure 2. The amino acid side chains whose mutations have a large effect on the RNA-binding properties of the complex are shown in red, while the amino acids that have a weaker effect are shown in orange. binding by closing over the nucleic acids. The sequence of the loop in M.jannaschii includes four positively charged residues and is disordered in the crystal structure. To examine the role of the B4–B5 loop in nucleic acid binding, we have created a 5 ‘loop mutant’ in which Arg155, Lys156 and Arg157 were mutate to alanine, serine and alanine, respectively. Based on the same criteria of surface exposure and sequence con- servation, a further set of residues were identiﬁed in the N-terminal domain of the E subunit, which folds into a trun- 10 cated RNP domain. These comprised Lys33, Arg37, Lys40 and Asp41. Figure 4 shows the position of the mutated residues on the crystal structure of the M.jannaschii E/F complex. The expres- sion levels and purity of the mutants were comparable with 15 that of the wild-type protein. Moreover, all mutants were thermostable (75 C) and behaved in a similar manner to the wild-type heterodimer on gel ﬁltration, demonstrating that the structural integrity of the mutant E/F complexes were not compromised by the amino-acid substitutions. The two excep- 20 tions were the mutants involving Lys40 and Asp41, which could not be puriﬁed to the same standards and were therefore not tested for binding. The RNA-binding properties of the mutants were then compared with those of the wild-type pro- tein. Examples of RNA-binding experiments for a selection of 25 mutants are reported in Figure 5A and B, while all the results are summarized in Figure 5C. While mutation of Phe95, Phe98 and Ser111 only mildly affect the interaction with the nucleic acids, most of the mutants showed a decrease in RNA binding when compared with the wild-type protein. The loop mutant, as well as muta- 30 tion of a number of positively charged residues (Lys33, Arg37, Lys152 and Lys160), virtually abolish RNA binding. When the residues whose mutation has have a strong effect in the interaction are displayed on the crystal structure of the M.jannaschii E/F heterodimer (Figure 4), a putative RNA- 35 binding path emerges involving both the N-terminal and C-terminal domains. Dissecting the RNA-binding region in the human Rpb4/Rpb7 complex We applied a similar approach to study the RNA-binding 40 properties of the human Rpb4/Rpb7 complex. The sequence identity between subunit E and Rpb7 is only 21% of the amino acids that are conserved in the S1 motif of the archaeal homologue, only Phe 98 is also well conserved in the euka- ryotic homologues. However, a similar pattern of exposed, 45 conserved residues in the C-terminal domain of the eukaryotic Rpb7 subunits can be inferred. These residues include Thr90, Asn93, Lys94, Phe98, Phe107, Ser109, His111, Arg151, Asp153 and Phe158. Another set of conserved, surface- exposed residues can be found in the N-terminal domain, 50 comprising His14, Glu33 and Lys41. These residues were mutated to either alanine, lysine or glutamic acid and are shown on Figure 4. As was the case with the E/F mutant complexes described above, the expression levels and purity of the human Rpb4/ 55 Rpb7 mutants were comparable with the wild-type protein. Unfortunately we have been unable to express any protein with a mutation at position 98; attempts to change the phenylalan- ine residue to either alanine or glutamic acid seriously affected the solubility of the protein. The RNA-binding properties of 60 the mutants were then compared with those of the wild-type protein. Examples of RNA-binding experiments for a selection of mutants are reported in Figure 5D and E, while all the results are summarized in Figure 5F. As in the archaeal protein, residues can be classiﬁed into 65 two categories, based on the impact of the mutation on the RNA-binding properties. Thr90, Asn93, Lys94 and Phe107 decrease the binding by about a factor of two, while the other residues have a stronger impact on the interaction. When the residues which have a strong effect are displayed 70 on the crystal structure of the human Rpb4/Rpb7 heterodimer, a pattern similar to that observed in the archaeal system emerges, with the putative RNA-binding path spanning both domains of the Rpb7 subunit. DISCUSSION 75 In the yeast 12-subunit RNAPII complex, the Rpb4/Rpb7 het- erodimer is positioned with the Rpb7 subunit orientated towards the 10-subunit core so that the Rpb4 helices are on the outside of the complex (Figure 6) (16). The large insertion between helices H1 and H2 in yeast Rpb4 is therefore posi- 80 tioned on the outer face of the polymerase molecule, well away from the core subunits. However, the very variable N-terminal segment of Rpb4 is in close proximity to the polymerase core. The 13 N-terminal amino acids of human Rpb4 are not conserved in eukaryotes and are disordered in the crystal 85 structure. In contrast, the solvent exposed Glu14 and Glu15 in