Several crystal forms of the Bacillus stearothermophilus 50 S ribosomal particles

A kinetic analysis is presented of proton translocation, TMPD+ formation and oxidation of endogenous respiratory carriers during oxygen pulses of TMPD supplemented rat-liver mitochondria. The results show that antimycin-insensitive proton ejection observed under coupled conditions derives from oxidation of endogenous respiratory carriers and re-reduction of TMPD+ by hydrogenated donors and not from proton pumping by cytochrome oxidase as claimed by other investigators. The observations presented provide an example of certain interpretative difficulties in the use of redox mediators and of the methodological approaches that can be used to avoid these.     

Crystallization of 70 S ribosomes and 30 S ribosomal subunits from Thermus thermophilus

Well-ordered three-dimensional crystals of 70 S ribosomes and 30 S ribosomal subunits from extremely thermophilic bacteria Thermus thermophilus have been obtained. Positively stained thin sections of the crystals have been analyzed by electron microscopy. Redissolved crystalline ribosomes and small ribosomal subunits reveal sedimentation constants of 70 S and 30 S, respectively, and are functionally active in the poly(U)-system.     

E. coli HflX interacts with 50S ribosomal subunits in presence of nucleotides

HflX is a GTP binding protein of unknown function. Based on the presence of the hflX gene in hflA operon, HflX was believed to be involved in the lytic-lysogenic decision during phage infection in Escherichia coli. We find that E. coli HflX binds 16S and 23S rRNA - the RNA components of 30S and 50S ribosomal subunits. Here, using purified ribosomal subunits, we show that HflX specifically interacts with the 50S. This finding is in line with the homology of HflX to GTPases involved in ribosome biogenesis. However, HflX-50S interaction is not limited to a specific nucleotide-bound state of the protein, and the presence of any of the nucleotides GTP/GDP/ATP/ADP is sufficient. In this respect, HflX is different from other GTPases. While E. coli HflX binds and hydrolyses both ATP and GTP, only the GTP hydrolysis activity is stimulated by 50S binding. This work uncovers interesting attributes of HflX in ribosome binding.     

Modification of 50S ribosomal subunits withN-bromosuccinimide

The 50S subunits ofEscherichia coli ribosomes were modified with the tryptophan reagentN-bromosuccinimide, and the sulfhydryl groups, the modification of which is accompanied by stimulation of polypeptide synthesis (López-Rivas, A. et al. (1978) Eur. J. Biochem. 92, 121), were regenerated by incubation with simple thiols. This treatment inactivates poly(U)-dependent polyphenylalanine synthesis, peptidyl transferase and elongation factor G-dependent GTPase. Incubation with proteins from untreated 70S ribosomes produces partial reactivation of polyphenylalanine synthesis and GTPase activity. Modification is accompanied by loss of 4–5 tryptophan residues per subunit.Abbreviation SucNBr N-bromosuccinimide     

Characterization of Escherichia coli 50S ribosomal protein L31

The published C-terminal sequence of Escherichia coli 50S ribosomal protein L31, ellipsisRFNK (Brosius, J. (1978) Biochemistry 17, 501-508), differs from that predicted by the gene sequence, ellipsisRFNKRFNIPGSK (GenBank accession no. X78541). This discrepancy might be due to post-translational processing of the protein. To examine this possibility, we have isolated L31 from E. coli strain MRE600 and sequenced the C-terminal tryptic peptide. We find the sequence to be FBIPGSK. Size comparisons of L31 from several E. coli strains demonstrate that all are identical in size to the protein isolated from MRE600 and larger than the previously described protein, indicating that ellipsisRFNKRFNIPGSK represents the true C-terminus of L31. In addition, we show that the failure to identify L31 in many ribosome preparations is probably due to the protein's loose association with the ribosome and its ability to form various intramolecular disulfide bonds, leading to L31 forms with distinct mobilities in gels.     

Negatively stained 50 S ribosomal subunits of Escherichia coli

Ribosomes are large nucleoproteins of approximately 3 X 10(6) Mr. In contrast to helical or spherical nucleoproteins (viruses) of similar size (which consist of several hundred small asymmetric units reproduced by symmetry), ribosomes are completely asymmetric; therefore, the amount of structural information (defined by the number of independent image elements) necessarily increases from about 10 to 20 to about 1000 to 2000 (at resolutions of the order of 2 nm). With present techniques, only stained single particles can be studied in the electron microscope. Our published work on the 30 S subunit and on the 50 S subunit has demonstrated that three-dimensional reconstructions of stained single particles show a great number of structural details that are reproducible if the particles have the same orientation. One of the main results of this paper is the final proof of this reproducibility from detailed comparisons of individual 50 S subunits and of independent averages over a few (3 to 5) particles in the kidney or crown orientation; in the latter case, even after a chemical modification. The 50 S subunit is non-uniformly stained along the optical axis. It displays a complicated, stain-filled channel-like structure, within which is approximately the partial volume expected for the RNA. The particle shows an irregular but reproducible boundary surface against the stain. At several sites, the channel structure protrudes to the surface. Since the secondary structure of the RNA is well known, one might try to locate it in the subunit after chemical identification of its surface contacts (the 3' end of 23 S RNA and the 3' end of the 5 S RNA have been localized). Most interesting is a groove on the surface, which might accommodate the mRNA.     

Reversible modification of 50S ribosomal subunits with dimethylmaleic anhydride

Summary The reversible modification of protein amino groups with dimethylmaleic anhydride, which had already been used to dissociate proteins from the 70S ribosomes of Escherichia coli (Pintor-Toro, J. A., et al. (1979) Biochemistry 18, 3219) was applied to the preparation of protein-deficient particles from the 50S subunits. Three successive cycles of treatment with dimethylmaleic anhydride, separation of dissociated proteins and regeneration of the modified amino groups produce partially inactivated ribosomal cores lacking proteins L7, L11 and L12, and having very small amounts of L1, L6 and L10. Incubation of these cores with the corresponding split proteins is accompanied by complete reactivation of the polypeptide synthesizing activity as compared with control 50S subunits.Abbreviation DMMA 2,3-dimethylmaleic anhydride     

Nuclear export and cytoplasmic maturation of ribosomal subunits

Based on the characterization of ribosome precursor particles and associated trans-acting factors, a biogenesis pathway for the 40S and 60S subunits has emerged. After nuclear synthesis and assembly steps, pre-ribosomal subunits are exported through the nuclear pore complex in a Crm1- and RanGTP-dependent manner. Subsequent cytoplasmic biogenesis steps of pre-60S particles include the facilitated release of several non-ribosomal proteins, yielding fully functional 60S subunits. Cytoplasmic maturation of 40S subunit precursors includes rRNA dimethylation and pre-rRNA cleavage, allowing 40S subunits to achieve translation competence. We review current knowledge of nuclear export and cytoplasmic maturation of ribosomal subunits.     

Characteristic views of E. coli and B. stearothermophilus 30S ribosomal subunits in the electron microscope

Large sets of electron microscopic images of the 30S ribosomal subunits of Bacillus stearothermophilus (914 molecules) and Escherichia coli (422 molecules) were analysed with image processing techniques. Using computer alignment and a new multivariate statistical classification scheme, three predominant views of the subunit were found for both species. These views, which together account for approximately 90% of the population of images, were determined to a reproducible resolution of up to 1.7 nm, thus elucidating many new structural details. The angular spread of the molecular orientations around the three main stable positions is remarkably small (less than 8 degrees). Some of the current models for the small ribosomal subunit are incompatible with our new results.     

Structural insights into methyltransferase KsgA function in 30S ribosomal subunit biogenesis

The assembly of the ribosomal subunits is facilitated by ribosome biogenesis factors. The universally conserved methyltransferase KsgA modifies two adjacent adenosine residues in the 3'-terminal helix 45 of the 16 S ribosomal RNA (rRNA). KsgA recognizes its substrate adenosine residues only in the context of a near mature 30S subunit and is required for the efficient processing of the rRNA termini during ribosome biogenesis. Here, we present the cryo-EM structure of KsgA bound to a nonmethylated 30S ribosomal subunit. The structure reveals that KsgA binds to the 30S platform with the catalytic N-terminal domain interacting with substrate adenosine residues in helix 45 and the C-terminal domain making extensive contacts to helix 27 and helix 24. KsgA excludes the penultimate rRNA helix 44 from adopting its position in the mature 30S subunit, blocking the formation of the decoding site and subunit joining. We suggest that the activation of methyltransferase activity and subsequent dissociation of KsgA control conformational changes in helix 44 required for final rRNA processing and translation initiation.     

Structural heterogeneity of 30S and 50S ribosomal subunits in Bacillus licheniformis

Proteins of free and polysome-derived ribosomal subunits of Bacillus licheniformis were fractionated by means of two-dimensional polyacrylamide gel electrophoresis. The free and derived 30S subunits have twelve protein components in common. Five proteins are present on the free 30S subunits only, whereas four proteins are exclusively found on the derived 30S subunits. The free and derived 50S subunits have at least twenty-eight proteins in common. Four proteins are unique for the derived and one for the free 50S subunits.     

Incorporation of the yeast mitochondrial ribosomal protein Mrp2 into ribosomal subunits requires the mitochondrially encoded Var1 protein

Mrp2 is a protein component of the small subunit of mitochondrial ribosomes in the yeast Saccharomyces cerevisiae. We have examined the expression of Mrp2 in yeast mutants lacking mitochondrial DNA and found that the steady-state level of Mrp2 is dramatically decreased relative to wild type. These data suggest that the accumulation of Mrp2 depends on the expression of one or more mitochondrial gene products. The mitochondrial genome of S. cerevisiae encodes two components of the small ribosomal subunit, 15S rRNA and the Var1 protein, both of which are necessary for the formation of mature 37S subunits. Several studies have shown that in the absence of Var1 incomplete subunits accumulate, which lack a limited number of ribosomal proteins. Here, we show that Mrp2 is one of the proteins absent from subunits lacking Var1, indicating that Var1 plays an important role in the incorporation of Mrp2 into mitochondrial ribosomal subunits.     

The effect of mutated mitochondrial ribosomal proteins S16 and S22 on the assembly of the small and large ribosomal subunits in human mitochondria

Mutations in mitochondrial small subunit ribosomal proteins MRPS16 or MRPS22 cause severe, fatal respiratory chain dysfunction due to impaired translation of mitochondrial mRNAs. The loss of either MRPS16 or MRPS22 was accompanied by the loss of most of another small subunit protein MRPS11. However, MRPS2 was reduced only about 2-fold in patient fibroblasts. This observation suggests that the small ribosomal subunit is only partially able to assemble in these patients. Two large subunit ribosomal proteins, MRPL13 and MRPL15, were present in substantial amounts suggesting that the large ribosomal subunit is still present despite a non-functional small subunit.