6442 Nucleic Acids Research, 2005, Vol. 33, No. 19 Figure 6. Ribbon diagram showing the location of the mutations and their effect on RNA binding, within the context of the RNAPII. The crystal structure of the entire 12-subunits RNAPII from yeast (PDB entry: 1wcm) is shown, with the 10 core subunits colour-coded as in Cramer et al. (12). The Rpb4 and Rpb7 subunits are shown on the left in pink and blue, respectively. The yeast residues structurally equivalent to the residues that have been mutated in the human Rpb7 are shown in red and yellow. As in Figure 4, residues that have a strong effect on RNA binding are shown in red, while orange indicates residues that have a weaker effect on binding. ssRNA in vitro. Despite a high degree of sequence similarity, no residue is absolutely conserved throughout the entire motif. From a structural viewpoint, the motif folds into a 5-stranded b-barrel known as OB fold, a common and ubiquitous struc- 5 tural motif, with no conserved sequence pattern, often involved in oligonucleotide binding. To date, no direct struc- tural information is available on the interaction of S1 domains with nucleic acids, but the structures of several OB folds have been determined in the presence of RNA or DNA (23). 10 Although the speciﬁc details of the interactions differ, in all these proteins oligonucleotide binding occurs on the face of the barrel containing the conserved residues in the S1 motif. Gel mobility shift assays have conﬁrmed that the yeast Rpb4/Rpb7 heterodimer binds single-stranded nucleic acid, 15 with an apparent dissociation constant of 0.7 mM for DNA and 1.2 mM for RNA (17). We have subsequently demon- strated that both the archaeal E/F complex and the yeast A14/A43 complex (its homologue in RNAPI) are able to bind nucleic acid with comparable afﬁnities (24). This indic- 20 ates that the RNA-binding function is evolutionary conserved in the archaeal and eukaryotic RNA polymerases. We have now quantiﬁed the binding of RNA to both the archaeal E/F heterodimer and the human Rpb4/Rpb7 heterodimer and in both cases determined a dissociation constant of 400–500 nM. 25 This relatively low-binding afﬁnity is consistent with the pro- posed role of the complex in the non-speciﬁc, transient binding of the newly synthesized RNA transcript. We have used the structure of both the archaeal M.jannaschii E/F complex and the human Rpb4/Rpb7 complex to identify residues potentially 30 important for RNA recognition. In the crystal structure of the M.jannaschii subunit, no elec- tron density is visible for part of the loop connecting strand B4 and B5. In a number of OB folds this loop has been shown to play a central role in DNA or RNA binding (using arginine, serine and lysine residues to interact with the sugar-phosphate 35 backbone) and often undergoes a conformational change to trap the nucleic acid (23). The sequence of the loop in M.jannaschii includes four positively charged residues that may play a role in non-sequence-speciﬁc nucleic acid binding. Although the type and order of these residues are not con- 40 served in archaeal sequences, charged and polar residues are usually present. We have created a triple mutant in the E subunit, in which three positively charged residues (Arg155, Lys156 and Arg157) were eliminated. The mutation of the loop has a remarkable effect, completely abolishing 45 RNA binding. Mutating two adjacent positively charged resi- dues (Lys152 and Lys160) to glutamic acids also completely abolishes RNA binding. In the human Rpb7 sequence the loop is shorter, but mutation of the surrounding residues (Arg151, Asp153 and Phe158) also nearly abolish RNA binding, con- 50 ﬁrming the importance of this region in protein–nucleic acid interaction. In deletion mutagenesis studies of S.cerevisiae Rpb7 (17), a mutant lacking the B4–B5 loop was shown to retain nucleic acid binding but was found to be inactive in transcription. 55 Mutants in this earlier study were designed based on the homo- logy to the S1 motif, without knowledge of the actual struc- ture. The B4–B5 loop deletion (residues 151–158) included part of b-strands B4 and B5, but the fact that such deletion had no effect on nucleic acid binding may be correlated to the lack 60 of positively charged residues in this region of the yeast sequence. The S1 motif in the C-terminal domain of subunit E was the primary candidate for mutagenesis studies. However, the topo- logy of the N-terminal domain of subunit E/Rpb7 resembles a 65 ‘truncated’, non-canonical RNP fold. RNP domains are the most widely found and best characterized ssRNA-binding motifs and fold into a b1 a1 b2 b3 a2 b4 structure, with the four antiparallel b-strands packing against the two helices. A truncated RNP motif, lacking a2 and b4, is present 70 in the structure, and resembles most closely the anticodon- binding domain of phenylalanine tRNA synthetase (25). How- ever it was not clear whether this was simply a structural module, or whether it may be involved in RNA binding. When the criteria for mutagenesis were applied to the 75 N-terminal domain, only a handful of residues for mutagenesis could be identiﬁed. A number of well-conserved residues are located on two loops at the bottom of this domain (loop A1–K2 and A3–A4; Figure 2), including a number of proline and glycine residues, as well as hydrophobic amino acids. How- 80 ever the crystal structure of the 12-subunit complex shows that these loops are involved in the interaction with the RNAP core, in particular with helix 1 of Rpb6, three short regions of Rpb1 (including the N-terminus and the linker leading to the CTD) and the anchor region of Rpb2 (16). 85 The conserved residues are therefore not available for RNA binding within the polymerase context, and have not there- fore been tested. No reasonably conserved, surface-exposed residue could be identiﬁed on the canonical RNA-binding surface of the RNP domain. However, a few residues that 90 fulﬁlled the criteria for mutagenesis were found on the face of the RNP domain which includes helix K2, and were shown to have an effect on RNA binding.