Translocation of tRNA during protein synthesis.

Coupled translocation of tRNA and mRNA in the ribosome during protein synthesis is one of the most challenging and intriguing problems in the field of translation. We highlight several key questions regarding the mechanism of translocation, and discuss possible mechanistic models in light of the recent crystal structures of the ribosome and its subunits.

The path of messenger RNA through the ribosome.

Using X-ray crystallography, we have directly observed the path of mRNA in the 70S ribosome in Fourier difference maps at 7 A resolution. About 30 nucleotides of the mRNA are wrapped in a groove that encircles the neck of the 30S subunit. The Shine-Dalgarno helix is bound in a large cleft between the head and the back of the platform. At the interface, only about eight nucleotides (-1 to +7), centered on the junction between the A and P codons, are exposed, and bond almost exclusively to 16S rRNA. The mRNA enters the ribosome around position +13 to +15, the location of downstream pseudoknots that stimulate -1 translational frame shifting.

Spontaneous erythromycin resistance mutation in a 23S rRNA gene, rrlA, of the extreme thermophile Thermus thermophilus IB-21.

Spontaneous, erythromycin-resistant mutants of Thermus thermophilus IB-21 were isolated and found to carry the mutation A2058G in one of two 23S rRNA operons. The heterozygosity of these mutants indicates that A2058G confers a dominant or codominant phenotype in this organism. This mutation provides a valuable tool for the genetic manipulation of the 23S rRNA genes of Thermus.

Streptomycin-resistant and streptomycin-dependent mutants of the extreme thermophile Thermus thermophilus.

We have isolated spontaneous streptomycin-resistant, streptomycin-dependent and streptomycin-pseudo-dependent mutants of the thermophilic bacterium Thermus thermophilus IB-21. All mutant phenotypes were found to result from single amino acid substitutions located in the rpsL gene encoding ribosomal protein S12. Spontaneous suppressors of streptomycin dependence were also readily isolated. Thermus rpsL mutations were found to be very similar to rpsL mutations identified in mesophilic organisms. This similarity affords greater confidence in the utility of the crystal structures of Thermus ribosomes to interpret biochemical and genetic data obtained with Escherichia coli ribosomes. In the X-ray crystal structure of the T. thermophilus HB8 30 S subunit, the mutated residues are located in close proximity to one another and to helices 18, 27 and 44 of 16 S rRNA. X-ray crystallographic analysis of ribosomes from streptomycin-resistant, streptomycin-pseudo-dependent and streptomycin-dependent mutants described here is expected to reveal fundamental insights into the mechanism of tRNA selection, translocation, and conformational dynamics of the ribosome.

Crystal structure of the ribosome at 5.5 A resolution.

We describe the crystal structure of the complete Thermus thermophilus 70S ribosome containing bound messenger RNA and transfer RNAs (tRNAs) at 5.5 angstrom resolution. All of the 16S, 23S, and 5S ribosomal RNA (rRNA) chains, the A-, P-, and E-site tRNAs, and most of the ribosomal proteins can be fitted to the electron density map. The core of the interface between the 30S small subunit and the 50S large subunit, where the tRNA substrates are bound, is dominated by RNA, with proteins located mainly at the periphery, consistent with ribosomal function being based on rRNA. In each of the three tRNA binding sites, the ribosome contacts all of the major elements of tRNA, providing an explanation for the conservation of tRNA structure. The tRNAs are closely juxtaposed with the intersubunit bridges, in a way that suggests coupling of the 20 to 50 angstrom movements associated with tRNA translocation with intersubunit movement.

Construction of low-resolution x-ray crystallographic electron density maps of the ribosome.

Advances in X-ray crystallography now allow biological macromolecules of almost any size to be imaged at atomic resolution. Here, I outline the strategy that allowed for the solution of the 70S ribosome structure to 7.8-A resolution. The most important factors involve the effective use of synchrotron radiation and the application of existing crystallographic software to very large structures.

The location of protein S8 and surrounding elements of 16S rRNA in the 70S ribosome from combined use of directed hydroxyl radical probing and X-ray crystallography.

Ribosomal protein S8, which is essential for the assembly of the central domain of 16S rRNA, is one of the most thoroughly studied RNA-binding proteins. To map its surrounding RNA in the ribosome, we carried out directed hydroxyl radical probing of 16S rRNA using Fe(II) tethered to nine different positions on the surface of protein S8 in 70S ribosomes. Hydroxyl radical-induced cleavage was observed near the classical S8-binding site in the 620 stem, and flanking the other S8-footprinted regions of the central domain at the three-helix junction near position 650 and the 825 and 860 stems. In addition, cleavage near the 5' terminus of 16S rRNA, in the 300 region of its 5' domain, and in the 1070 region of its 3'-major domain provide information about the proximity to S8 of RNA elements not directly involved in its binding. These data, along with previous footprinting and crosslinking results, allowed positioning of protein S8 and its surrounding RNA elements in a 7.8-A map of the Thermus thermophilus 70S ribosome. The resulting model is in close agreement with the extensive body of data from previous studies using protein-protein and protein-RNA crosslinking, chemical and enzymatic footprinting, and genetics.

X-ray crystal structures of 70S ribosome functional complexes.

Structures of 70S ribosome complexes containing messenger RNA and transfer RNA (tRNA), or tRNA analogs, have been solved by x-ray crystallography at up to 7.8 angstrom resolution. Many details of the interactions between tRNA and the ribosome, and of the packing arrangements of ribosomal RNA (rRNA) helices in and between the ribosomal subunits, can be seen. Numerous contacts are made between the 30S subunit and the P-tRNA anticodon stem-loop; in contrast, the anticodon region of A-tRNA is much more exposed. A complex network of molecular interactions suggestive of a functional relay is centered around the long penultimate stem of 16S rRNA at the subunit interface, including interactions involving the "switch" helix and decoding site of 16S rRNA, and RNA bridges from the 50S subunit.

Identification of an RNA-protein bridge spanning the ribosomal subunit interface.

The 7.8 angstrom crystal structure of the 70S ribosome reveals a discrete double-helical bridge (B4) that projects from the 50S subunit, making contact with the 30S subunit. Preliminary modeling studies localized its contact site, near the bottom of the platform, to the binding site for ribosomal protein S15. Directed hydroxyl radical probing from iron(II) tethered to S15 specifically cleaved nucleotides in the 715 loop of domain II of 23S ribosomal RNA, one of the known sites in 23S ribosomal RNA that are footprinted by the 30S subunit. Reconstitution studies show that protection of the 715 loop, but none of the other 30S-dependent protections, is correlated with the presence of S15 in the 30S subunit. The 715 loop is specifically protected by binding free S15 to 50S subunits. Moreover, the previously determined structure of a homologous stem-loop from U2 small nuclear RNA fits closely to the electron density of the bridge.

A specific monovalent metal ion integral to the AA platform of the RNA tetraloop receptor.

Metal ions are essential for the folding and activity of large catalytic RNAs. While divalent metal ions have been directly implicated in RNA tertiary structure formation, the role of monovalent ions has been largely unexplored. Here we report the first specific monovalent metal ion binding site within a catalytic RNA. As seen crystallographically, a potassium ion is coordinated immediately below AA platforms of the Tetrahymena ribozyme P4-P6 domain, including that within the tetraloop receptor. Interference and kinetic experiments demonstrate that potassium ion binding within the tetraloop receptor stabilizes the folding of the P4-P6 domain and enhances the activity of the Azoarcus group I intron. Since a monovalent ion binding site is integral to the tetraloop receptor, a tertiary structural motif that occurs frequently in RNA, monovalent metal ions are likely to participate in the folding and activity of a wide diversity of RNAs.

Complementary sets of noncanonical base pairs mediate RNA helix packing in the group I intron active site.

Helix packing is critical for RNA tertiary structure formation, although the rules for helix-helix association within structured RNAs are largely unknown. Docking of the substrate helix into the active site of the Tetrahymena group I ribozyme provides a model system to study this question. Using a novel chemogenetic method to analyze RNA structure in atomic detail, we report that complementary sets of noncanonical base pairs (a G.U wobble pair and two consecutively stacked sheared A.A pairs) create an RNA helix packing motif that is essential for 5'-splice site selection in the group I intron. This is likely to be a general motif for helix-helix interaction within the tertiary structures of many large RNAs.

A magnesium ion core at the heart of a ribozyme domain.

Large ribozymes require divalent metal ions to fold. We show here that the tertiary structure of the Tetrahymena group I intron P4-P6 domain nucleates around a magnesium ion core. In the domain crystal structure, five magnesium ions bind in a three-helix junction at the centre of the molecule. Single atom changes in any one of four magnesium sites in this three-helix junction destroy folding of the entire 160-nucleotide P4-P6 domain. The magnesium ion core may be the RNA counterpart to the protein hydrophobic core, burying parts of the RNA molecule in the native structure.

RNA structure: crystal clear?

Structured RNAs play an essential role in chromosome maintenance, RNA processing, protein biosynthesis, and protein transport. To understand RNA function in these diverse biological systems, the rules for RNA folding and recognition must be learned. Recent crystal structures of hammerhead ribozymes, a group I intron domain, and RNA duplexes provide new insights into the principles of RNA folding and function.

Metal-binding sites in the major groove of a large ribozyme domain.

BACKGROUND: Group I self-splicing introns catalyze sequential transesterification reactions within an RNA transcript to produce the correctly spliced product. Often several hundred nucleotides in size, these ribozymes fold into specific three-dimensional structures that confer activity. The 2.8 A crystal structure of a central component of the Tetrahymena thermophila group I intron, the 160-nucleotide P4-P6 domain, provides the first detailed view of metal binding in an RNA large enough to exhibit side-by-side helical packing. The long-range contacts and bound ligands that stabilize this fold can now be examined in detail. RESULTS: Heavy-atom derivatives used for the structure determination reveal characteristics of some of the metal-binding sites in the P4-P6 domain. Although long-range RNA-RNA contacts within the molecule primarily involve the minor groove, osmium hexammine binds at three locations in the major groove. All three sites involve G and U nucleotides exclusively; two are formed by G.U wobble base pairs. In the native RNA, two of the sites are occupied by fully-hydrated magnesium ions. Samarium binds specifically to the RNA by displacing a magnesium ion in a region critical to the folding of the entire domain. CONCLUSIONS: Bound at specific sites in the P4-P6 domain RNA, osmium (III) hexammine produced the high-quality heavy-atom derivative used for structure determination. These sites can be engineered into other RNAs, providing a rational means of obtaining heavy-atom derivatives with hexammine compounds. The features of the observed metal-binding sites expand the known repertoire of ligand-binding motifs in RNA, and suggest that some of the conserved tandem G.U base pairs in ribosomal RNAs are magnesium-binding sites.

Crystal structure of a group I ribozyme domain: principles of RNA packing.

Group I self-splicing introns catalyze their own excision from precursor RNAs by way of a two-step transesterification reaction. The catalytic core of these ribozymes is formed by two structural domains. The 2.8-angstrom crystal structure of one of these, the P4-P6 domain of the Tetrahymena thermophila intron, is described. In the 160-nucleotide domain, a sharp bend allows stacked helices of the conserved core to pack alongside helices of an adjacent region. Two specific long-range interactions clamp the two halves of the domain together: a two-Mg2+-coordinated adenosine-rich corkscrew plugs into the minor groove of a helix, and a GAAA hairpin loop binds to a conserved 11-nucleotide internal loop. Metal- and ribose-mediated backbone contacts further stabilize the close side-by-side helical packing. The structure indicates the extent of RNA packing required for the function of large ribozymes, the spliceosome, and the ribosome.

RNA tertiary structure mediation by adenosine platforms.

The crystal structure of a group I intron domain reveals an unexpected motif that mediates both intra- and intermolecular interactions. At three separate locations in the 160-nucleotide domain, adjacent adenosines in the sequence lie side-by-side and form a pseudo-base pair within a helix. This adenosine platform opens the minor groove for base stacking or base pairing with nucleotides from a noncontiguous RNA strand. The platform motif has a distinctive chemical modification signature that may enable its detection in other structured RNAs. The ability of this motif to facilitate higher order folding provides one explanation for the abundance of adenosine residues in internal loops of many RNAs.