6436 Nucleic Acids Research, 2005, Vol. 33, No. 19 Figure 1. Structure based sequence alignment of the Rpb4 and Rpb7 subunits. Sequences of the human (HS), yeast (SC) and M.jannaschii (MJ) orthologues of Rpb4 and Rpb7 are included. The secondary structure of the human protein determined using DSSP (33) is indicated above the sequences while secondary structural elements in the yeast (PDB entry 1y14) and M.jannaschii (PDB entry 1go3) proteins are indicated by boxes. Amino acids not present in the high-resolution crystal structures are indicated by italics. Residues which are well conserved in archaeal and eukaryotic sequences are highlighted using a green background. In the Rpb7 alignment, a red background indicates residues mutagenized in this study which have a strong effect on the RNA-binding properties of the protein whereas an orange background indicates residues whose mutation has a weaker effect on RNA binding. Residues in strands B1–B6 of Rpb7 share sequence similarity with proteins containing an S1 motif and form a b-barrel structure called an OB fold. determined by crystallography at 1.75 s resolution (13). The crystal structure of the 12-subunit yeast RNAPII complex was initially determined at 4.2 s resolution (14,15). As the low- resolution map did not allow an independent atomic model to 5 be built for the eukaryotic Rpb4/Rpb7 complex, the structure of the archaeal E/F dimer was simply placed into the electron density. More recently the crystal structure of the S.cerevisiae Rpb4/Rpb7 complex has been determined (16), which has allowed the 12-subunit RNAPII complex to be reﬁned to 10 3.8 s resolution. The archaeal subunit E (the Rpb7 homologue) has an elongated two-domain structure, containing two potential single-stranded RNA (ssRNA)-binding motifs: a truncated RNP motif at the N-terminus and a S1 motif at the 15 C-terminus (13). The presence of an S1 motif in the sequence and structure suggests that its role is to bind single-stranded nucleic acids. Indeed, the eukaryotic Rpb4/Rpb7 complex has been shown to bind to both single-stranded DNA (ssDNA) and ssRNA with comparable afﬁnities (17). Although proteins 20 containing an OB fold can bind either ssRNA or ssDNA, the particular subgroup containing the sequence signature of the S1 motif bind ssRNA without sequence speciﬁcity. We therefore proposed a model for the function of the heterodimer, in which the S1 motif of Rpb7 interacts with the nascent RNA transcript, possibly assisted by the truncated 25 RNP domain (13). We have now determined the crystal structure of the human Rpb4/Rpb7 complex. To gain insight into the contributions of different regions of the heterodimer to RNA binding, we have carried out structure-based mutational analysis of conserved, 30 surface-exposed amino acid residues in both the archaeal E/F and the human Rpb4/Rpb7 protein complex and tested the ability of each mutant protein to bind RNA. We show that residues within an elongated patch on the surface of human Rpb7 contribute to RNA binding. The RNA-binding region 35 includes residues forming the OB fold in the C-terminal domain of the protein as well as additional residues from the N-terminal domain. In the context of the yeast 12-subunit RNAPII complex, the RNA-binding region of human Rpb4/ Rpb7 corresponds to the face of the heterodimer adjacent to 40 the 10-subunit core. We also show that, in spite of relatively weak sequence identity, residues within a surface patch in a corresponding position on the surface of subunit E of the archaeal polymerase also contribute to RNA binding.