Meanderings of the mRNA through the ribosome.

A map of how mRNA travels through the ribosome is critical for any detailed understanding of the process of translation. This feat has recently been achieved using X-ray crystallography. The structure reveals, for the first time, details of the interactions between the mRNA and the 30S subunit beyond those at the tRNA binding sites. Elements of both 16S rRNA and ribosomal proteins contribute to mRNA binding. This work also identifies two tunnels that the mRNA passes through as it wraps around the 30S subunit. The mechanisms and mechanics of reading frame selection, translational fidelity, and translocation can now be informed by the structure.

In vitro reconstitution of 30S ribosomal subunits using complete set of recombinant proteins.

This system allows convenient purification of large quantities of all of the small subunit ribosomal proteins by overexpression from cloned genes. This not only allows large-scale reconstitution of 30S subunits from individual proteins, but also facilitates protein purification greatly. These proteins can be reconstituted into functional 30S subunits using an ordered assembly protocol based on the in vitro 30S assembly map. Reconstitution of 30S subunits using this system enables mutant or modified proteins, such as Fe(II)-BABE-derivatized proteins, to be incorporated into subunits for studying ribosome structure and function.

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.

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.

Efficient reconstitution of functional Escherichia coli 30S ribosomal subunits from a complete set of recombinant small subunit ribosomal proteins.

Previous studies have shown that the 30S ribosomal subunit of Escherichia coli can be reconstituted in vitro from individually purified ribosomal proteins and 16S ribosomal RNA, which were isolated from natural 30S subunits. We have developed a 30S subunit reconstitution system that uses only recombinant ribosomal protein components. The genes encoding E. coli ribosomal proteins S2-S21 were cloned, and all twenty of the individual proteins were overexpressed and purified. Reconstitution, following standard procedures, using the complete set of recombinant proteins and purified 16S ribosomal RNA is highly inefficient. Efficient reconstitution of 30S subunits using these components requires sequential addition of proteins, following either the 30S subunit assembly map (Mizushima & Nomura, 1970, Nature 226:1214-1218; Held et al., 1974, J Biol Chem 249:3103-3111) or following the order of protein assembly predicted from in vitro assembly kinetics (Powers et al., 1993, J MoI Biol 232:362-374). In the first procedure, the proteins were divided into three groups, Group I (S4, S7, S8, S15, S17, and S20), Group II (S5, S6, S9, Sll, S12, S13, S16, S18, and S19), and Group III (S2, S3, S10, S14, and S21), which were sequentially added to 16S rRNA with a 20 min incubation at 42 degrees C following the addition of each group. In the second procedure, the proteins were divided into Group I (S4, S6, S11, S15, S16, S17, S18, and S20), Group II (S7, S8, S9, S13, and S19), Group II' (S5 and S12) and Group III (S2, S3, S10, S14, and S21). Similarly efficient reconstitution is observed whether the proteins are grouped according to the assembly map or according to the results of in vitro 30S subunit assembly kinetics. Although reconstitution of 30S subunits using the recombinant proteins is slightly less efficient than reconstitution using a mixture of total proteins isolated from 30S subunits, it is much more efficient than reconstitution using proteins that were individually isolated from ribosomes. Particles reconstituted from the recombinant proteins sediment at 30S in sucrose gradients, bind tRNA in a template-dependent manner, and associate with 50S subunits to form 70S ribosomes that are active in poly(U)-directed polyphenylalanine synthesis. Both the protein composition and the dimethyl sulfate modification pattern of 16S ribosomal RNA are similar for 30S subunits reconstituted with either recombinant proteins or proteins isolated as a mixture from ribosomal subunits as well as for natural 30S subunits.

Probing the rRNA environment of ribosomal protein S5 across the subunit interface and inside the 30 S subunit using tethered Fe(II).

A newly developed 30 S subunit reconstitution system using a complete set of recombinant proteins was used to study the ribosomal RNA (rRNA) neighborhood of ribosomal protein S5 in 30 S subunits and 70 S ribosomes by directed hydroxyl radical probing. Using three cysteine-containing mutant S5 proteins derivatized with 1-(p-bromoacetamidobenzyl)-Fe(II)-EDTA, we expanded on experiments carried out earlier using a natural protein reconstitution system. Natural 16 S rRNA, Fe(II)-S5, and the other recombinant ribosomal proteins were reconstituted into 30 S subunits. Both 30 S subunits and 70 S ribosomes containing Fe(II)-S5 were purified, and hydroxyl radicals were generated in situ from the tethered Fe(II). In 30 S subunits, 16 S rRNA nucleotides targeted by two positions on S5, C21 and C99, were virtually identical to those observed in the previous work, supporting the validity of the recombinant protein reconstitution system for probing studies. Interestingly, new cleavages were detected using Fe(II)-C129-S5, possibly reflecting incorporation of more derivatized protein into 30 S subunits due to the increased reconstitution efficiency of the recombinant protein system. These newly targeted positions overlap, but are distinct from, those observed using Fe(II) tethered to C21, which is near C129 in the S5 structure. In 70 S ribosomes, the cleavage pattern of 16 S rRNA was very similar to that observed in 30 S subunits for all target sites except for the absence of those at the extreme 5' end of 16 S rRNA. Additionally, probing of 70 S ribosomes from Fe-C99-S5 results in cleavage of 23 S rRNA in the 1690-1770 region of domain IV. These data provide constraints for the three-dimensional location of nucleotides within domain IV of 23 S ribosomal RNA relative to known features of the 30 S subunit.

Directed hydroxyl radical probing of 16S ribosomal RNA in ribosomes containing Fe(II) tethered to ribosomal protein S20.

The 16S ribosomal RNA neighborhood of ribosomal protein S20 has been mapped, in both 30S subunits and 70S ribosomes, using directed hydroxyl radical probing. Cysteine residues were introduced at amino acid positions 14, 23, 49, and 57 of S20, and used for tethering 1-(p-bromoacetamidobenzyl)-Fe(II)-EDTA. In vitro reconstitution using Fe(II)-derivatized S20, together with the remaining small subunit ribosomal proteins and 16S ribosomal RNA (rRNA), yielded functional 30S subunits. Both 30S subunits and 70S ribosomes containing Fe(II)-S20 were purified and hydroxyl radicals were generated from the tethered Fe(II). Hydroxyl radical cleavage of the 16S rRNA backbone was monitored by primer extension. Different cleavage patterns in 16S rRNA were observed from Fe(II) tethered to each of the four positions, and these patterns were not significantly different in 30S and 70S ribosomes. Cleavage sites were mapped to positions 160-200, 320, and 340-350 in the 5' domain, and to positions 1427-1430 and 1439-1458 in the distal end of the penultimate stem of 16S rRNA, placing these regions near each other in three dimensions. These results are consistent with previous footprinting data that localized S20 near these 16S rRNA elements, providing evidence that S20, like S17, is located near the bottom of the 30S subunit.

A 2'-phosphotransferase implicated in tRNA splicing is essential in Saccharomyces cerevisiae.

The last step of tRNA splicing in the yeast Saccharomyces cerevisiae is catalyzed by an NAD-dependent 2'-phosphotransferase, which transfers the splice junction 2'-phosphate from ligated tRNA to NAD to produce ADP-ribose 1"-2" cyclic phosphate. We have purified the phosphotransferase about 28,000-fold from yeast extracts and cloned its structural gene by reverse genetics. Expression of this gene (TPT1) in yeast or in Escherichia coli results in overproduction of 2'-phosphotransferase activity in extracts. Tpt1 protein is essential for vegetative growth in yeast, as demonstrated by gene disruption experiments. No obvious binding motifs are found within the protein. Several candidate homologs in other organisms are identified by searches of the data base, the strongest of which is in Schizosaccharomyces pombe.

tRNA splicing in yeast and wheat germ. A cyclic phosphodiesterase implicated in the metabolism of ADP-ribose 1",2"-cyclic phosphate.

Adenosine diphosphate (ADP)-ribose 1",2"-cyclic phosphate (Appr > p) is produced as a result of transfer RNA (tRNA) splicing in the yeast Saccharomyces cerevisiae and probably in other eukaryotes. Endonucleolytic cleavage and ligation result in a mature length tRNA with a 2'-phosphate at the splice junction. This 2'-phosphate is transferred to NAD to produce Appr > p. Metabolism of Appr > p requires hydrolysis of the 1",2"-cyclic phosphate linkage. We show here that yeast has a unique cyclic phosphodiesterase that can hydrolyze Appr > p, ribose 1,2-cyclic phosphate, and ribose 1,3-cyclic phosphate to the corresponding ribose 1-phosphate derivatives. The cyclic phosphodiesterase is highly specific for Appr > p; there is 20-fold less activity on ribose 1,3-cyclic phosphate and no detectable activity on nucleoside 2',3'-cyclic phosphates. A similar cyclic phosphodiesterase is present in wheat germ. The wheat germ cyclic phosphodiesterase activity co-chromatographs with a 2',3'-cyclic nucleotide 3'-phosphodiesterase that was previously identified and purified. The purified wheat germ enzyme has a distinct preference for Appr > p and ribose cyclic phosphate compared to guanosine 2',3'-cyclic phosphate and shares other biochemical characteristics with the yeast enzyme.

An NAD derivative produced during transfer RNA splicing: ADP-ribose 1"-2" cyclic phosphate.

Transfer RNA (tRNA) splicing is essential in Saccharomyces cerevisiae as well as in humans, and many of its features are the same in both. In yeast, the final step of this process is removal of the 2' phosphate generated at the splice junction during ligation. A nicotinamide adenine dinucleotide (NAD)-dependent phosphotransferase catalyzes removal of the 2' phosphate and produces a small molecule. It is shown here that this small molecule is an NAD derivative: adenosine diphosphate (ADP)-ribose 1"-2" cyclic phosphate. Evidence is also presented that this molecule is produced in Xenopus laevis oocytes as a result of dephosphorylation of ligated tRNA.

Renal carcinoma--unusual presentation and angiographic findings.

A case of papillary renal cell carcinoma with clinical and angiographic features of an abscess is reported. Papillary renal carcinomas constitute 14% of renal adenocarcinomas. These tumors have slower growth rate and better prognosis.

Mediastinal lymphangioma.

Mediastinal lymphangioma is an uncommon benign tumor accounting for 0.7 to 4.5% of all mediastinal masses. Lymphangiomas consist of dilated cystic lymph spaces lined by single layers of endothelium and do not undergo malignant change. Most mediastinal lymphangiomas are asymptomatic. There are no specific radiological findings. Surgical excision is the treatment of choice and the prognosis is excellent.

Renal carbuncle: simulation of tumor response to epinephrine.

A case of renal carbuncle with unusual angiographic findings is presented. The abscess showed abnormal vessels on selective angiogrpahy which were enhanced after intra-arterial epinephrine. New foci of abnormal vessels were also seen on the postepinephrine angiogram.