fragment of its mRNA, has been determined and compared with the structure of L1–rRNA complex. The comparison revealed a network of strongly conserved H-bonds, which are inaccessible to the solvent. Atoms involved in these H- bonds must be responsible for the speciﬁc RNA–protein recog- nition. MATERIALS AND METHODS Identification of a MjaL1mRNA fragment suitable for MjaL1–mRNA complex formation The ﬁrst aim was to design a ‘minimal’ mRNA fragment, an RNA fragment as short as possible which still would retain the full afﬁnity for L1. From the structure of the L1–rRNA complex (5), we concluded that, apart from the nucleotides essential for L1 recognition (shown with black background in Figure 1), the two helices ﬂanking the asymmetric loop might be essential for L1 binding. Comparison of the L1 binding sites on the mRNA from M.jannaschii with that of the closely related Figure 1. Secondary structure of the regulatory L1-binding site on the L1mRNA of M.jannaschii and derivatives thereof used in binding experiments and crystallization trials. (A) Localization of the L1-binding site and of the 30S ribosomal subunit as part of the translation initiation complex on the L1mRNA. Nucleotides different in the M.vannielii L1mRNA are shown with smaller sized letters. (B) Fragment MjaL1mRNA-49 comprising nucleotides +28 to +68 with four additional base pairs (italic) at the 30 and the 50 end. Fragment MjaL1mRNA-38a comprising nucleotides +24 to +38 and +54 to +72 is closed by the tetraloop UUGC (italic). Fragment MjaL1mRNA-38b is similar to MjaL1mRNA-38a, but the free 30 and 50 ends have been replaced by an ex- tended helix as in MjaL1mRNA-49. Fragment MjaL1mRNA-30 comprising nucleotides +28 to +38 and +54 to +68 is closed by a tetraloop as in MjaL1mRNA-38. The cluster of nucleotides conserved in all 23S rRNA and mRNA binding sites is shown with black background. Nucleic Acids Research, 2005, Vol. 33, No. 2 479 M.vannielii revealed that the 7 bp helix (nucleotides +28 to +34 and +62 to +68) ﬂanking the asymmetric loop was identical in both species, whereas the sequence of the upper part of the stem–loop structure (comprising nucleotides +40 to +51) was only poorly conserved between the two species (Figure 1A). Thus, it was a reasonable assumption that this stem–loop structure was not essential for L1 binding. Several RNA constructs that included the regulatory MjaL1 binding site were synthesized (Figure 1B) and tested for their MjaL1-binding activity using ﬁlter binding assays. As Table 1 shows, fragments MjaL1mRNA-49, MjaL1mRNA-38a and MjaL1mRNA-38b (Figure 1B) bind L1 with virtually the same (full) afﬁnity as a 250 nt MjaL1mRNA fragment, con- ﬁrming that the stem–loop structure distal to the asymmetric loop does not contribute to the L1–mRNA interaction, and can be shortened to a 3 bp helix closed by a tetraloop without any loss of afﬁnity for L1. Unexpectedly, the 30 nt fragment MjaL1mRNA-30 (Figure 1B) does not exhibit any speciﬁc afﬁnity for MjaL1, indicating that the 7 bp helix alone is not sufﬁcient for L1 binding. The apparent dissociation constant determined for MjaL1 in complex with the MjaL1mRNA-30 (Kd of 10 7 M) is virtually the same as for MjaL1 in complex with a 16S rRNA fragment, which was used as a negative control. MjaL1, in general, shows a rather high unspeciﬁc afﬁnity for any RNA. L1 from the closely related M.vannielii, which does not show such an unspeciﬁc afﬁnity for RNA, does not bind to MjaL1mRNA-30, but exhibits the full afﬁnity for the other three short mRNA fragments tested (data not shown). All three transcripts exhibiting the full afﬁnity for L1 were used to prepare MjaL1–mRNA complexes for crystallization; so far the best crystals were obtained with the 49 nt fragment. Large single crystals of this complex containing SeMetL1 were used for the data collection and for structural studies. Protein and RNA preparation The fragment MjaL1mRNA-49 was obtained by transcription from linearized plasmid DNA using T7 RNA polymerase. RNA was puriﬁed on denaturing (8 M urea) 15% (w/v) acryl- amide (19:1, acrylamide/bis-acrylamide) gels, using 90 mM Tris-borate, pH 8.2, 1 mM EDTA, as running buffer. RNA was eluted by 50 mM Tris–HCl buffer, pH 7.5 (25 C), 1 mM EDTA, puriﬁed by anion-exchange (DEAE-Sepharose) chromatography, precipitated by ethanol and dissolved in 1 mM sodium citrate, pH 6.4. The gene for MjaL1 was cloned and overexpressed in E.coli strain BL21(DE3) as a host (12). To avoid the potential misincorporation of amino acids Table 1. Binding of MjaL1 to different RNA fragments RNA MjaL1mRNA-250 MjaL1mRNA-49 MjaL1mRNA-38a MjaL1mRNA-38b MjaL1mRNA-30 Mja16SrRNA Description 250 nt fragment of the MjaL1mRNA (22), comprising nucleotides 29 to +220, used as positive control Figure 1B Figure 1B Figure 1B Figure 1B 145 nt fragment of 16S rRNA (23) used as negative control Kd [M] 5.0 · 10 10 5.2 · 10 10 5.8 · 10 10 4.9 · 10 10 1.0 · 10 7 2.5 · 10 7 Binding was carried out in the presence of 350 mM KCl as described in Materials and Methods.