478–485 Nucleic Acids Research, 2005, Vol. 33, No. 2 doi:10.1093/nar/gki194 Ribosomal protein L1 recognizes the same specific structural motif in its target sites on the autoregulatory mRNA and 23S rRNA Natalia Nevskaya, Svetlana Tishchenko, Azat Gabdoulkhakov, Ekaterina Nikonova, 1 Oleg Nikonov, Alexei Nikulin, Olga Platonova , Maria Garber, Stanislav Nikonov and Wolfgang Piendl1,* Institute of Protein Research, Russian Academy of Sciences, 142290 Pushchino, Moscow region, Russia and 1Innsbruck Medical University, Biocentre, Fritz-Prengl-Str.3, A-6020 Innsbruck, Austria Received November 23, 2004; Revised and Accepted December 24, 2004 DDBJ/EMBL/GenBank accession no. 1U63 ABSTRACT The RNA-binding ability of ribosomal protein L1 is of profound interest since the protein has a dual function as a ribosomal protein binding rRNA and as a trans- lational repressor binding its mRNA. Here, we report the crystal structure of ribosomal protein L1 in com- plex with a specific fragment of its mRNA and compare it with the structure of L1 in complex with a specific fragment of 23S rRNA determined earlier. In both com- plexes, a strongly conserved RNA structural motif is involved in L1 binding through a conserved network of RNA–protein H-bonds inaccessible to the solvent. These interactions should be responsible for specific recognition between the protein and RNA. A large number of additional non-conserved RNA–protein H-bonds stabilizes both complexes. The added con- tribution of these non-conserved H-bonds makes the ribosomal complex much more stable than the regulatory one. INTRODUCTION L1, one of the largest ribosomal proteins, is located on the side protuberance, opposite the L7/L12 stalk of the 50S ribosomal subunit. In the isolated state L1 proteins have two different conformations: a closed conformation in the case of bacterial L1 from Thermus thermophilus (1) and an open one in the case of its archaeal homologues from Methanococcus jannaschii (2) or Methanococcus thermolithotrophicus (3). L1 is a primary RNA-binding ribosomal protein, which associates independently, speciﬁcally and strongly with 23S rRNA (4). We have recently determined the crystal structure of L1 in complex with a speciﬁc 55 nt fragment of 23S rRNA (5). It is known that L1 proteins in Escherichia coli and in Methanococcus species regulate their own expression by bind- ing to their mRNAs, thereby acting as translational repressors (6–10). L1 from Escherichia coli (EcoL1) mediates auto- genous regulation of translation by binding to a region within the leader sequence, close to the Shine–Dalgarno sequence, of the mRNA of the L11 operon coding for ribosomal proteins L1 and L11 (6). L1 from M.vannielii (MvaL1) was shown to be an auto-regulator of the MvaL1 operon encoding ribosomal proteins L1, L10 and L12 (8). It was also shown that EcoL1 can inhibit the in vitro translation of MvaL1 polycistronic mRNA and, conversely, that MvaL1 can inhibit the synthesis of both L11 and L1 proteins of E.coli (9). However, there are no structural data available on the conformation of the mRNA to which L1 binds, either in isolation or with L1 bound. In bacteria and archaea, the L1 regulatory target site exhibits high similarity in both sequence and secondary struc- ture to the L1 binding site on the 23S rRNA. L1 proteins from mesophilic and thermophilic bacteria and archaea bind to the speciﬁc site on 23S rRNA with 5- to 10-fold higher afﬁnity than to their regulatory binding site on the mRNA (10). This difference ﬁts the requirements of classical regulation of ribosomal synthesis (feedback inhibition) based on direct competition between the two binding sites. Direct comparison of the structures of the complexes formed by regulatory ribo- somal proteins with their targets on rRNA and mRNA is an attractive task. Only one such investigation has been made recently for ribosomal protein S8 (11). We prepared and crystallized several complexes between bacterial and archaeal L1 proteins and speciﬁc fragments of their mRNAs (S. Tishchenko, unpublished). The structure of one of these complexes, M.jannaschii L1 (MjaL1) with a *To whom correspondence should be addressed: Tel: +43 512 507 3531; Fax: +43 512 507 2872; Email: Wolfgang.Piendl@uibk.ac.at 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. For commercial re-use permissions, please contact journals.permissions@oupjournals.org. ª 2005, the authors Nucleic Acids Research, Vol. 33 No. 2 ª Oxford University Press 2005; all rights reserved