Figure 4. Stereo ribbon representation of the L1–mRNA and L1–rRNA complexes in the same orientation. helix 78, similar to the structure formed by the two perpen- dicular helices of the mRNA binding site. In both RNAs, the junction of the two helices contains nucleotides strictly con- served in all L1-binding sites in large rRNAs as well as in sites speciﬁc for L1 in mRNAs from those bacteria and archaea, for which feedback regulation has been experimentally proved. These nucleotides are connected by a network of conserved hydrogen bonds, most of which are inaccessible to the solvent. This network strongly stabilizes the unique three-dimensional structure of the region. Comparison of these unique sites in both kinds of L1–RNA complexes shows that they are struc- turally conserved with an r.m.s. deviation between P atoms of 0.50 s. Comparison of the protein–RNA interactions in the complexes In the ribosomal and regulatory complexes, protein L1 inter- acts with RNA through both domains. Domain I contacts RNA via the slightly concave surface formed by the inner face of the b-sheet and two spatially adjacent loops, containing residues identical in all known L1 sequences. The number of residues that contact the RNA is substantially less in domain II than in domain I, particularly in the L1–mRNA complex. The contact area between L1 protein and RNA is more extensive in the ribosomal complex than in the regulatory one due to differences in the spatial organization of the two RNAs. In each complex there are two sites of interaction with Nucleic Acids Research, 2005, Vol. 33, No. 2 483 L1. In the mRNA, one site includes mainly nucleotides of the ﬁrst helix and of the junction region. On the rRNA surface, the analogous site is formed by nucleotides of helix 77 and one strand of helix 78. Both RNA molecules interact through this site mainly with domain I of the protein. Highly conserved nucleotides of this site (G33, G34, G36, C63 in the mRNA or G2124, G2125, G2127, C2174 in the rRNA) form hydrogen bonds with strictly conserved residues located in the beginning of strand b1 and spatially adjacent loop b9–b10. The second region of interaction is formed by nucleotides of loop B in the rRNA or the interconnecting loop in the mRNA. Nucleotides of this site interact with L1 residues of domain II. The more than 2-fold shortening of the interconnecting loop in the mRNA relative to loop B in the rRNA decreases signiﬁcantly the number of RNA–protein contacts in the regulatory complex. It should be noted that nucleotides at 30 and 50 ends, which form 4 bp in the ﬁrst helix of MjaL1mRNA-49 fragment, are not involved in interaction with L1. Consequently, we can suggest that the absence of L1-binding capacity in the 30 nt fragment of MjaL1mRNA may be associated with some deformation of the unique RNA structure in this short mRNA fragment. Probably, these 4 bp are needed for retention of this unique spatial structure of L1-binding site on RNA. RNA–protein recognition Binding sites on the protein and RNA surfaces include regions responsible for RNA–protein recognition (we call them ‘recognition modules’) and regions, which form additional intermolecular contacts (21). It is known that many ribosomal proteins are structurally and functionally interchangeable between ribosomes from different species. These data suggest that the structure of recognition modules on the proteins and rRNAs should be highly conserved and complementary to each other. The polar atoms of recognition modules usually form a network of intermolecular RNA–protein hydrogen bonds, some of which are inaccessible to the solvent. We have recently suggested that atoms involved in these hydrogen bonds are responsible for RNA–protein recognition (21). Detailed analysis of all known crystal structures of L1 pro- teins from different organisms revealed a structurally invariant region in domain I. This region contains a cluster of strictly conserved amino acid residues; their relative positions and side-chain conformations are stabilized by intramolecular hydrogen bonds. It is noteworthy that there is no difference in the conformation of this region in the isolated L1 molecule and in L1 bound to rRNA or mRNA (Figure 5A). It seems that the structure of this site is preformed to bind RNA and does not undergo sufﬁcient conformational changes upon binding. These data enable us to consider this region of the protein as its RNA recognition module. It should be mentioned that we suggested a key role of this region in RNA binding previously (3), based solely on the analysis of crystal structures of isolated L1 proteins. The comparison between the two L1–RNA com- plexes proves this suggestion to be correct. In two L1–RNA complexes, the rRNA and mRNA frag- ments also possess practically identical structures in and around the junction of two corresponding helices (Figure 5B). These regions contain highly conserved nucleotides and form the unique conformation strongly stabilized by intramolecular hydrogen bonds.