Thermus flavus 5S rRNA with a molecular weight of about 40 kDa was modified at the 5' and 3' ends. Crystals were obtained under earth and microgravity conditions. The best crystals were obtained during NASA space mission STS 94. For the first time, it was possible to collect a complete data set from 5S rRNA crystals to 7.8 A resolution and to assign the space group as R32, with unit-cell parameters a = b = 110.3, c = 387.6 A, alpha = beta = 90, gamma = 120 degrees.

The ribosomal 5S RNA is approximately 120 nucleotides long and is an integral part of the large ribosomal subunit. Several parts of the 5S rRNA interact specifically with several ribosomal proteins [1-3]. Nevertheless its precise role in protein synthesis remains unclear. It is clear that reconstituted 50S ribosomal subunits, lacking the 5S rRNA, are inactive in proteinbiosynthesis [4], Structural studies will support a more derailed understanding of the 5S rRNA function. Our extensive attempts to crystallise ribosomal 5S RNA have led to crystals, which diffract to about 7.5 Angstrom [5]. For the investigation of some special features like GU basepairs and hairpin-loops in the ribosomal 5S RNA from Thermus flavus we have divided the 5S rRNA into five domains [6] (A to E) and have solved three crystal structures (Figure 1): Helix I of domain A has been determined at 2.4 Angstrom [7]. Helix V of domain E without the terminal hairpin-loop has been determined at 1.6 Angstrom and Helix V of domain E including the terminal hairpin-loop has been determined at 3.0 Angstrom [8]. Furthermore it was possible to observe a general mode of intermolecular interaction, which occur only in RNA structures and it was possible to demonstrate the structural importance of water molecules for the stability of RNA-molecules.

The synthetic RNA fragment 5′‐CUGGGCGG(GCGA)CCGCCUGG (nucleotides in parentheses indicate the loop region) corresponds to the natural sequence of domain E from nucleotides 79–97 of the Thermus flavus 5S rRNA including a hairpin loop. The RNA structure determined at 3.0 Å and refined to an R ‐value of 24.1% also represents the first X‐ray structure GNRA tetraloop. The loop is in distinctly different conformation from other GNRA tetraloops analyzed by NMR. The conformation of the two molecules in the asymmetric unit is influenced and stabilized by specific intermolecular contacts. The structural features presented here give evidence for the ability of RNA molecules to adapt to specific environments.

MACROMOLECULESthe orphaned 3U-tem1inal end in the minor groove of the adjacent duplex where it synm1etrically pairs with the 5'-terminal guanine to form a d[G*(G.C)] base-tliplet.Our findings extend to the minor groove a DNA hydrogen bonding pattem which permits basepair recognition dming homologous recombination.(I l ACCGGTACCGGT P4 3 , (40.2, 40.2, 57.6 A). !, 2.8 A (2) ACCGACGTCGGT Pl,(40.5,40.1.40.5A, 82.6.116.2.80.7°),3,2.6A(3) ACCGACGTCGGT R3, (63.1, 63.1, 44.5 A), 1, 2.1 A (4) ACCGACGruCGGT Pl. (40.3,40.3.40.2 A, 87.1,87.3,68.0°),3,3.2A(5) CCGACCGACGTCGGTCG, R3, (100.7,100.7, 79.8 A), 3. S.OA

The ribosomal protein L36 of the thermophilic bacterium Thermus thermophilus was separated by a newly developed analytical method of Reversed Phase Chromatography and subsequently gas phase sequenced without cleavage in one single step. The complete sequence of the 37 amino acids, containing a zinc-binding motive, is presented. Furthermore, the N-terminal sequences of L5, L9, L18, L24, L32, L33 and L35 from Thermus thermophilus are shown.

The ribosomal 5S RNA is an essential constituent of the large ribosomal subunit. To overcome the difficulties of crystallizing large RNA molecules such as 5S rRNAs, we decided to divide the 5S rRNA in five domains A through E to determine their structure. Recently we determined the crystal structural of the helical domain A. Here we report the crystallization of the chemically synthesized domain E of the Thermus flavus 5S rRNA. The crystal form is trigonal with unit cell dimensions: a = b = 42.80 A and c = 162.20 A. Diffraction-data to 2.8 A have been recorded and the structure solution is currently underway by means of MIR and MAD techniques.

The ribosomal 5S RNA is an essential constituent of the large ribosomal subunit. To overcome the difficulties of crystallizing large RNA molecules such as 5S rRNAs, we decided to divide the 5S rRNA in five domains A through E to determine their structure. Recently we determined the crystal structural of the helical domain A. Here we report the crystallization of the chemically synthesized domain E of the Thermus flavus 5S rRNA. The crystal form is trigonal with unit cell dimensions: and . Diffraction‐data to 2.8 Å have been recorded and the structure solution is currently underway by means of MIR and MAD techniques.

This is the first high resolution crystal structure of an RNA molecule made by solid phase chemical synthesis and representing a natural RNA. The structure of the domain A of Thermus flavus ribosomal 5S RNA is refined to R = 18% at 2.4 A including 159 solvent molecules. Most of the 2'-hydroxyl groups as well as the phosphate oxygens are involved either in specific hydrogen bonds in intermolecular contacts or to solvent molecules. The two U-G and G-U base-pairs are stabilized by H-bonds supplied via three water molecules to compensate for the lack of base-pair hydrogen bonds. The structure shows for the first time in detail the importance of highly ordered internal water in stabilizing an RNA structure.

Crystals of domain A of Thermus flavus 5S rRNA have been obtained. The space group was found to be P43 with unit-cell dimensions a = b = 30.10 and c = 86.80 Å. Data to 2.3 Å have been recorded and solution of the structure is currently underway by means of molecular-replacement techniques.

Crystals of domain A of Thermus flavus 5S rRNA have been obtained. The space group was found to be P4(3) with unit-cell dimensions a = b = 30.10 and c = 86.80 A. Data to 2.3 A have been recorded and solution of the structure is currently underway by means of molecular-replacement techniques.

Lead ions have been applied to the structural analysis of 5S rRNA from Thermus thermophilus, Bacillus stearothermophilus and Escherichia coli. Based on the distribution of Pb(II)-induced cleavages, some minor modifications of the consensus secondary structure model of 5S rRNA are proposed. They include the possible base pairing between nucleotides at positions 11 and 109, as well as changes in secondary interactions within the helix B region. The 'prokaryotic arm' region is completely resistant to hydrolysis in the three RNA species, suggesting that it is a relatively stable, highly ordered structure. Hydrolysis of E. coli 5S rRNA complexed with ribosomal protein L18 shows, besides the shielding effect of the bound protein, a highly enhanced cleavage between A108 and A109. It supports the concept that the major L18-induced conformational change involves the junction of helices A, B and D.

Different stable forms of Escherichia coli and rat liver 5S rRNA have been probed by Pb(II)-induced hydrolysis. In the native A forms of 5S rRNA, Pb2+ reveal single-stranded RNA stretches and regions of increased conformational flexibility or distorted by the presence of bulged nucleotides. Hydrolysis of urea/EDTA-treated E. coli 5S rRNA (B form) shows the presence of two strong helical domains; helix A retained from the A form and a helix composed of RNA regions G33-C42 and G79-C88. Other RNA regions resistant to hydrolysis may be involved in alternative base pairing, causing conformational heterogeneity of that form. Pb(II)-induced hydrolysis distinguishes two different forms of rat liver 5S rRNA; the native A form and the form obtained by renaturation of 5S rRNA in the presence of EDTA. Pb(II)-hydrolysis data suggest that both forms are highly structured. In the latter form, the orientation of the bulged C66 is changed with respect to helix B. At the same time, a new helical segment is possibly formed, composed of nucleotides from helix C and loop c on one side and from helix E and loop d' on the other.

Crystals of purified 5 S rRNA from Thermus flavus have been obtained. The crystals diffract up to 8 Å resolution, using synchrotron radiation, and have the monoclinic space-group C2. The unit cell has the dimensions a = 190 Å, b = 110 Å, c = 138 Å and β = 117 °. The cell volume suggests the presence of four 5 S rRNA molecules per asymmetric unit.

The cytotoxin alpha-sarcin was employed to test the model of secondary and tertiary structures of plant 5S rRNAs, which we recently proposed [(1990) Int. J. Biol. Macromol. (in press)]. alpha-Sarcin is a novel ribonuclease that hydrolyzes phosphodiester bonds adjacent to purines in nucleic acids. The digestion pattern obtained for lupin and wheat germ 5S rRNAs strongly suggests the existence of tertiary interactions between residues C34, C35, C36, A37 and G85, G86, G87, U88 as previously proposed. The results on the secondary structure of plant 5S rRNA are in line with a previously proposed model.