The racemate of the RNA duplex r(CUGGGCGG).r(CCGCCUGG) from Thermus flavus 5S rRNA has been crystallized and examined by X-ray crystallography. The space group is P1(_) with approximate unit-cell parameters a = 26.5, b = 38.0, c = 45.4 Angstrom, alpha = 113.1, beta = 100.5, gamma = 93.3 degrees. The structure was solved by molecular replacement. There are four RNA duplexes in the unit cell. The crystal lattice consists of columns of RNA duplexes. The duplexes are stacked end-to-end and are stabilized by intermolecular base-stacking interactions. Within each column the L-duplexes and D-duplexes are stacked alternately. Every other duplex in each stack has two alternative conformations, approximately equally occupied, corresponding to molecules oriented in opposite directions. Neighbouring columns are related by the crystallographic centre of symmetry. The unit cell also contains approximately 250 ordered water molecules and six ordered calcium ions. A glycerol molecule is visible in the minor groove interacting with a guanosine residue.

The shape of free Thermus flavus 5 S rRNA in solution at 1.3 nm resolution is restored from synchrotron x-ray scattering data using an ab initio simulated annealing algorithm. The free 5 S rRNA is a bent elongated molecule displaying a compact central region and two projecting arms, similar to those of the tRNA. Ribosomal 5 S rRNA, an essential component of the ribosome, is approximately 120 nucleotides long. The ribosomal particles lacking the 5 S rRNA have a strongly reduced activity in protein synthesis (1-3), in particular, reduced peptidyl transferase activity. Because of its functional importance and the fact that the 5 S rRNA interacts specifically with several ribosomal proteins (4), it is of great interest to know the threedimensional structure or a reliable shape of this RNA molecule. Nearly 1000 different prokaryotic and eukaryotic 5 S rRNA sequences have been determined so far. The predicted secondary structure (5) is shown in Fig. 1. The size of the 5 S rRNA limits the possibility of its three-dimensional structure determination by nuclear magnetic resonance (NMR). Therefore, in the past, we tried to crystallize several 5 S rRNA species (6, 7). The crystals of an isolated 5 S rRNA suitable for the x-ray analysis were obtained particularly from the thermophilic bacterium Thermus flavus. These crystals, however, diffract only to about 0.75 nm resolution and are extremely sensitive to radiation, even under cryogenic conditions. The intrinsic flexibility of the whole 5 S rRNA molecule and small differences in the primary structure seem to influence significantly the quality of the crystals. The T. flavus 5 S rRNA was then divided into five domains, A through E, and these domains were chemically synthesized and crystallized for x-ray analysis (8, 9). In parallel, we began to analyze the shape of the entire 5 S rRNA in solution using small angle scattering, an established method of monitoring the low resolution structure of biological macromolecules (10). Recently, new approaches have been developed to ab initio restore low resolution structure from the scattering data (11, 12), and these were successfully applied to study proteins and ribosomes (13-15). In the present paper, an ab initio low resolution model of the free 5 S rRNA in solution is derived from synchrotron radiation small angle x-ray scattering (SAXS) 1 data. The atomic models of the 5 S rRNA fragments are tentatively positioned inside the low resolution shape. The model obtained may be further used for crystallographic molecular replacement studies, as well as for the analysis of the complex formation with binding proteins in solution. EXPERIMENTAL PROCEDURESIsolation and Purification-The Escherichia coli cells from strain MRE600 were grown at 37°C in a Luria broth medium. The 70 S ribosomes were isolated by previously described extraction and centrifugation steps (2). The intact ribosomes were dissociated into their 30 and 50 S subunits at magnesium concentrations below 5 mM (1). The 5 S rRNA was separated from the large ribosomal subunit b...

Nucleic acid molecules in the mirror image or l-con®guration are unknown in nature and are extraordinarily resistant to biological degradation. The identi®cation of functional l-oligonucleotides called Spiegelmers offers a novel approach for drug discovery based on RNA. The sequence r(CUGGGCGG)Ár(CCGCCUGG) was chosen as a model system for structural analysis of helices in the l-con®guration as the structure of the d-form of this sequence has previously been determined in structural studies of 5S RNA domains, in particular domain E of the Thermus¯avus 5S rRNA [Perbandt et al. (2001), Acta Cryst. D57, 219±224]. Unexpectedly, the results of crystallization trials showed little similarity between the d-and the l-forms of the duplex in either the crystallization hits or the diffraction performance. The crystal structure of this l-RNA duplex has been determined at 1.9 A Ê resolution with R work and R free of 23.8 and 28.6%, respectively. The crystals belong to space group R32, with unit-cell parameters a = 45.7, c = 264.6 A Ê . Although there are two molecules in the asymmetric unit rather than one, the structure of the l-form arranges helical pairs in a head-totail fashion to form pseudo-continuous in®nite helices in the crystal as in the d-form. On the other hand, the wobble-like GÁC + base pair seen in the D-RNA analogue does not appear in the l-RNA duplex, which forms a regular double-helical structure with typical Watson±Crick base pairing.

The structure of the RNA duplex r(CUGGGCGG).r(CCGCCUGG) has been determined at 1.6 A resolution and refined to a final R factor of 18.3% (R(free) = 24.1%). The sequence of the RNA fragment resembles domain E of Thermus flavus 5S rRNA. A previously undescribed wobble-like G.C base-pair formation is found. Owing to the observed hydrogen-bond network, it is proposed that the cytosine is protonated at position N3. The unusual base-pair formation is presumably strained by intermolecular interactions. In this context, crystal packing and particular intermolecular contacts may have direct influence on the three-dimensional structure. Furthermore, this structure includes two G.U wobble base pairs in tandem conformation, with the purines forming a so-called 'cross-strand G stack'.

A novel peptide was designed which possesses nanomolar affinity of less than 20 nM for streptavidin. Therefore it was termed Nano-tag and has been used as an affinity tag for recombinant proteins. The minimized version of the wild type Nano-tag is a seven-amino acid peptide with the sequence fMDVEAWL. The three-dimensional structure of wild type streptavidin in complex with the minimized Nano-tag was analyzed at atomic resolution of 1.15 A and the details of the binding motif were investigated. The peptide recognizes the same pocket of streptavidin where the natural ligand biotin is bound, but the peptide requires significantly more space than biotin. Therefore the binding loop adopts an "open" conformation in order to release additional space for the peptide. The conformation of the bound Nano-tag corresponds to a 3(10) helix. However, the analysis of the intermolecular interactions of the Nano-tag with residues of the binding pocket of streptavidin reveals astonishing similarities to the biotin binding motif. In principle the three-dimensional conformation of the Nano-tag mimics the binding mode of biotin. Our results explain why the use of the Nano-tag in fusion with recombinant proteins is restricted to their N-terminus and we describe the special significance of the fMet residue for the high affinity binding mode.