Biochem. J. (2014) 462, 373–384 (Printed in Great Britain) doi:10.1042/BJ20131474 373 Archaeal MBF1 binds to 30S and 70S ribosomes via its helix–turn–helix domain Fabian BLOMBACH* 1,2 , Helene LAUNAY† 1,3 , Ambrosius P. L. SNIJDERS‡, Violeta ZORRAQUINO*, Hao WU* 4 , Bart DE KONING*, Stan J. J. BROUNS*, Thijs J. G. ETTEMA§, Carlo CAMILLONI‖, Andrea CAVALLI‖, Michele VENDRUSCOLO‖, Mark J. DICKMAN‡, Lisa D. CABRITA†, Anna LA TEANA¶, Dario BENELLI**, Paola LONDEI**, John CHRISTODOULOU† 5 and John VAN DER OOST* 5 *Laboratory of Microbiology, Wageningen University, Wageningen 6703HB, The Netherlands †Institute of Structural and Molecular Biology, University College London (UCL) and Birkbeck College, University of London, London WC1E 6BT, U.K. ‡Department of Chemical and Process Engineering, University of Sheffield, Sheffield S1 3JD, U.K. §Department of Cell and Molecular Biology, Science for Life Laboratory, Uppsala University, Uppsala SE-75137, Sweden ‖Department of Chemistry, University of Cambridge, Cambridge CB2 1EW, U.K. ¶Dipartimento di Scienze della Vita e dell’Ambiente, Universit` a Politecnica delle Marche, Ancona 60131, Italy **Dipartimento Biotecnologie Cellulari ed Ematologia, Universit` a degli Studi di Roma “La Sapienza”, Roma 00161, Italy MBF1 (multi-protein bridging factor 1) is a protein containing a conserved HTH (helix–turn–helix) domain in both eukaryotes and archaea. Eukaryotic MBF1 has been reported to function as a transcriptional co-activator that physically bridges transcription regulators with the core transcription initiation machinery of RNA polymerase II. In addition, MBF1 has been found to be associated with polyadenylated mRNA in yeast as well as in mammalian cells. aMBF1 (archaeal MBF1) is very well conserved among most archaeal lineages; however, its function has so far remained elusive. To address this, we have conducted a molecular characterization of this aMBF1. Affinity purification of interacting proteins indicates that aMBF1 binds to ribosomal subunits. On sucrose density gradients, aMBF1 co-fractionates with free 30S ribosomal subunits as well as with 70S ribosomes engaged in translation. Binding of aMBF1 to ribosomes does not inhibit translation. Using NMR spectroscopy, we show that aMBF1 contains a long intrinsically disordered linker connecting the predicted N-terminal zinc-ribbon domain with the C-terminal HTH domain. The HTH domain, which is conserved in all archaeal and eukaryotic MBF1 homologues, is directly involved in the association of aMBF1 with ribosomes. The disordered linker of the ribosome-bound aMBF1 provides the N-terminal domain with high flexibility in the aMBF1–ribosome complex. Overall, our findings suggest a role for aMBF1 in the archaeal translation process. Key words: helix–turn–helix domain (HTH domain), multi- protein bridging factor 1 (MBF1), ribosome, Sulfolobus, transcription, translation, translation fidelity. INTRODUCTION Archaea and eukaryotes share a common set of proteins involved in genetic information processing (transcription, translation and replication), including several proteins containing HTH (helix– turn–helix) domains [1–3]. Most of these proteins carry out functions within the core transcription machinery in both archaea and eukaryotes. This includes the eukaryotic protein MBF1 (multi-protein bridging factor 1) that has been shown to act as a transcriptional co-activator, transmitting the signal from eukaryote-specific transcription factors to the core transcription machinery by physically bridging these factors with the TBP (TATA-box-binding protein) via the HTH domain of MBF1 [4–7]. Besides its characterized function as a transcriptional co- activator, previous studies suggest that eukaryotic MBF1 might be a moonlighting protein. In yeast, a frameshift mutation in the mbf1 sequence as well as deletion of the entire mbf1 gene have been shown to alter the rate of ribosomal frameshifting as well as the sensitivity of the strains to aminoglycoside antibiotics including paromomycin [8–10]. In addition, yeast MBF1 has been recently shown to co-purify with Pab1 [poly(A)-binding protein 1]. The interaction is sensitive to RNase treatment, suggesting that MBF1 is associated with polyadenylated mRNA [11]. Furthermore, yeast MBF1 binds directly to RNA via the less-conserved N-terminal domain [11]. Similarly, human MBF1 was also identified as an mRNA-binding protein in embryonic stem cells and HEK (human embryonic kidney)-293 cells [12,13]. When the first archaeal genomes became available, aMBF1 (archaeal MBF1) orthologues were identified on the basis of sequence homology encompassing the HTH domain [2]; however, aMBF1 has remained functionally uncharacterized ever since. The evolutionary conservation of TBP across all eukaryotes and archaea might suggest that TBP and aMBF1 also interact in archaea [5]. However, the fact that experimental investigations using chimaeric constructs bearing HTH domains originating Abbreviations: aIF6, archaeal translation initiation factor 6; aMBF1, archaeal MBF1; aMBF1-C, C-terminal domain of aMBF1; aMBF1-N, N-terminal domain of aMBF1; HTH, helix–turn–helix; MBF1, multi-protein bridging factor 1; PFG, pulsed-field gradient; TBP, TATA-box-binding protein. 1 These authors contributed equally to this work. 2 Present address: Research Department of Structural and Molecular Biology, Institute of Structural and Molecular Biology, University College London (UCL), London WC1E 6BT, U.K. 3 Present address: CNRS-UMR 8576-University of Lille1, Villeneuve d’Ascq, Lille FR-59655, France. 4 Present address: Cardiovascular Biology Research Program, Oklahoma Medical Research Foundation, Oklahoma City, OK 73104, U.S.A. 5 Correspondence may be addressed to either of these authors (email j.christodoulou@ucl.ac.uk or john.vanderoost@wur.nl). Co-ordinates and structure factors of the aMBF1 (archaeal multi-protein bridging factor 1) helix–turn–helix domain have been deposited in the PDB under code 2MEZ. The NMR assignment data have been deposited in the BMRB under accession number 19028. c  The Authors Journal compilation c  2014 Biochemical Society Biochemical Journal www.biochemj.org