Nucleic Acids Research, 2005, Vol. 33, No. 19 6435–6444 doi:10.1093/nar/gki945 Crystal structure and RNA binding of the Rpb4/Rpb7 subunits of human RNA polymerase II Hedije Meka, Finn Werner, Suzanne C. Cordell, Silvia Onesti and Peter Brick* Department of Biological Sciences, Imperial College, London SW7 2BW, UK 5 Received September 5, 2005; Revised and Accepted October 17, 2005 ABSTRACT The Rpb4 and Rpb7 subunits of eukaryotic RNA poly- merase II (RNAPII) form a heterodimer that protrudes from the 10-subunit core of the enzyme. We have 10 obtained crystals of the human Rpb4/Rpb7 hetero- dimer and determined the structure to 2.7 A˚ resolu- tion. The presence of putative RNA-binding domains on the Rpb7 subunit and the position of the hetero- dimer close to the RNA exit groove in the 12 subunit 15 yeast polymerase complex strongly suggests a role for the heterodimer in binding and stabilizing the nascent RNA transcript. We have complemented the structural analysis with biochemical studies direc- ted at dissecting the RNA-binding properties of the 20 human Rpb4/Rpb7 complex and that of the homo- logous E/F complex from Methanocaldococcus jannaschii. A number of conserved, solvent- exposed residues in both the human Rpb7 subunit and the archaeal E subunit have been modified by 25 site-directed mutagenesis and the mutants tested for RNA binding by performing electrophoretic mobility shift assays. These studies have identified an elongated surface region on the corresponding face of both subunit E and Rpb7 that is involved 30 in RNA binding. The area spans the nucleic acid binding face of the OB fold, including the B4–B5 loop, but also extends towards the N-terminal domain. INTRODUCTION In the eukaryotic RNA polymerase II (RNAPII), two subunits 35 (Rpb4/Rpb7) form a heterodimer that associate with the 10- subunit core of the enzyme (1). The stoichiometry of RNAPII puriﬁed from Saccharomyces cerevisiae cells is dependent on growth conditions: in optimally growing cells only 20% of puriﬁed material includes the Rpb4/Rpb7 heterodimer, while 40 in the post-log phase virtually all the polymerase molecules contain the 12 subunits. RNAPII puriﬁed from rpb4D cells not only lacks the corresponding subunit Rpb4, but also contains no detectable Rpb7 (2). Whereas in S.cerevisiae RPB7 is an essential gene, RPB4 is dispensable under optimal growth conditions, but becomes essential during heat or cold shock, 45 and under nutrient depletion. Overexpression of Rpb7 partly compensates for the rpb4D stress phenotypes and Rpb7 can interact with RNAPII independently of Rpb4, although this interaction is not very stable and can only be detected when the subunit is overexpressed (3,4). In the S.cerevisiae system, 50 Rpb4/Rpb7 is necessary for promoter-directed transcription initiation but dispensable for transcription elongation and ter- mination in vitro (2) and activated transcription in vivo (5). Rpb4 also recruits the CTD phosphatase Fcp1 (6). Both Rpb4 and Rpb7 have been shown to have additional speciﬁc func- 55 tions in gene expression, such as regulating transcription- coupled repair through the Rad26-dependent pathway (7), in efﬁcient mRNA export (8) and in transcription termination (9). The heterodimer seems therefore to play a role in the intricate network linking transcription, RNA processing and mRNA 60 export. Archaea contain a single RNAP enzyme with subunits which show considerable sequence homology to the subunits of the eukaryotic RNAPII. The sequences of the Rpb7 sub- units from eukaryotes and the homologous E subunits from 65 archaea are well conserved while the sequence similarity between the eukaryotic Rpb4 subunits and the archaeal F subunits is very much lower (Figure 1). The archaeal RNAP is fully capable of promoter-directed transcription without subunits E/F (10). It is interesting to note that the stimulatory 70 effect of the archaeal basal transcription factor TFE (homo- logous to eukaryotic TFIIEa) is dependent on the RNAP sub- units E/F, also suggesting a role of E/F for transcription initiation in the archaea (11). Since the heterogeneity in the yeast polymerase prepara- 75 tions caused by the variable stoichiometry of the Rpb4/Rpb7 heterodimer interfered with crystallization, crystals of RNAPII diffracting to high resolution were originally obtained for the 10-subunit core (12). However, the 3D structure of the com- plex between the archaeal homologues of E and F had been 80 *To whom correspondence should be addressed. Tel: +44 20 75947704; Fax: +44 20 75890191; Email: p.brick@imperial.ac.uk Correspondence may also be addressed to Silvia Onesti. Tel: +44 20 75947704; Fax: +44 20 75890191; Email: s.onesti@imperial.ac.uk The Author 2005. Published by Oxford University Press. All rights reserved. The online version of this article has been published under an open access model. Users are entitled to use, reproduce, disseminate, or display the open access version of this article for non-commercial purposes provided that: the original authorship is properly and fully attributed; the Journal and Oxford University Press are attributed as the original place of publication with the correct citation details given; if an article is subsequently reproduced or disseminated not in its entirety but only in part or as a derivative work this must be clearly indicated. 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