484 Nucleic Acids Research, 2005, Vol. 33, No. 2 Figure 5. Invariant regions in L1 proteins and in the rRNA and mRNA fragments. Conserved H-bonds are shown with dotted lines. (A) The location of the invariant structure on the surface of L1 proteins is shown in green on the MjaL1 model (right). Superposition of the corresponding regions in L1 proteins in isolated and RNA-bound forms is shown on the left. Isolated MjaL1 is in gray, MjaL1 complexed with mRNA in magenta and SacL1 complexed with rRNA in brown. (B) Superposition of the mRNA (blue) and rRNA (green) fragments; the conserved unique structure is outlined (right). In the L1–rRNA and L1–mRNA complexes, polar atoms of the RNAs and L1 invariant regions form ﬁve conserved RNA–protein hydrogen bonds inaccessible to the solvent (Figure 5B). These bonds are formed by the atoms of amino acid residues Ser25, Glu27, Thr204, Met205 and of nucleotides G33, G34, C63. We have made three point mutations, Thr204Gly, Met205Gly and Met205Asp, and characterized them with respect to their RNA-binding potency by a ﬁlter- binding assay. The analysis revealed that the above listed mutants have decreased rRNA-binding ability compared with the wild-type protein and do not bind the mRNA at all. Structural modelling shows that these mutant proteins lose one of the con- served hydrogen bonds with RNA. Thus, in both cases conserved RNA–protein hydrogen bonds play a crucial role in L1–RNA recognition and binding. However, the binding of mRNA to L1 turned out to be more sensitive to point mutations than the binding of rRNA. This may be the result of considerably fewer contacts between L1 and mRNA than between L1 and rRNA. ACKNOWLEDGEMENTS This work was supported by the Russian Academy of Sciences, the Russian Foundation for Basic Research (No. 03-04-48327 and No. 04-04-49634), the Program of RAS on Molecular and Cellular Biology and the Program of the RF President on support of outstanding scientific schools. The research of M.G. was supported in part by International Research Scholar’s award from the Howard Hughes Medical Institute. The research of W.P. was supported by the Austrian Science Fund (FWF, P17164-B10). Funding to pay the Open Access publication