Reconstitution of peptide bond formation with Escherichia coli 23S ribosomal RNA domains

It was recently demonstrated that peptide bond formation can occur using an Escherichia coli naked 23S ribosomal RNA without any of the ribosomal proteins. Here, the six domains of the 23S ribosomal RNA were individually synthesized and shown to be capable, when complexed together, of stimulating the reaction. Omission and addition experiments indicated that the activity could be reconstituted solely by domain V at a concentration 10 times higher than that of the intact 23S ribosomal RNA, whereas domain VI could enhance the activity in trans. These findings suggest that fragments of an RNA molecule have the ability to associate into a functional whole.     

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.     

The phosphorylation of protein S6 modulates the interaction of the 40 S ribosomal subunit with the 5'-untranslated region of a dictyostelium pre-spore-specific mRNA and controls its stability

AC914 mRNA, a pre-spore-specific mRNA that accumulates only in the post-aggregation stage of development, is transcribed constitutively as shown by nuclear run-off experiments and by fusing its promoter to the luciferase reporter gene. The same mRNA disappears quickly from disaggregated cells. If the 5'-untranslated region (5'UTR) of the constitutively expressed Actin 15 mRNA is substituted for the 5'UTR of AC914 mRNA, this can no longer be destabilized and accumulates both in growing and disaggregated cells. If the 5'UTR of AC914 mRNA is substituted for the 5'UTR of Actin 15 mRNA, the latter accumulates only in aggregated cells. Pactamycin, but not other inhibitors of protein synthesis, prevents AC914 mRNA from being destabilized in disaggregated cells, suggesting a role of 40 S subunits in the destabilization. This has been confirmed by using an in vitro system in which the in vivo stability of different mRNAs is reproduced. A protein kinase A-dependent phosphorylation of ribosomal protein S6 determines whether 40 S subunits are capable or not of destabilizing AC914 mRNA in the in vitro system.     

Nucleotide sequence of the gene for ribosomal protein S17 from Dictyostelium discoideum

The nucleotide sequence of the gene for the Dictyostelium homologue of eukaryotic ribosomal protein S17 has been assembled from cDNA and genomic DNA clones. The predicted primary structure of the S17 protein displays a similar level of sequence identity with its counterparts from higher eukaryotes (53%) as other Dictyostelium ribosomal proteins. Although Dictyostelium genes usually are organized in a rather simple manner, the rps17 gene harbors two introns. One of them, located immediately 3' from the ATG initiator codon, appears to be ubiquitously conserved in eukaryotic rps17 genes.     

Yeast proteins related to the p40/laminin receptor precursor are required for 20S ribosomal RNA processing and the maturation of 40S ribosomal subunits

Numerous studies have linked the overexpression of the Mr 37,000 laminin receptor precursor (37-LRP) to tumor cell growth and proliferation. The role of this protein in carcinogenesis is generally considered in the context of its putative role as a precursor for the Mr 67,000 high-affinity laminin receptor. Recent studies have shown that 37-LRP, also termed p40, is a component of the small ribosomal subunit indicating that it may be a multifunctional protein. The p40/37-LRP protein is highly conserved phylogenetically, and closely related proteins have been identified in species as evolutionarily distant as humans and the yeast, Saccharomyces cerevisiae. Yeast homologues of p40/37-LRP are encoded by a duplicated pair of genes, RPS0A and RPS0B. The Rps0 proteins are essential components of the 40S ribosomal subunit. Previous results have shown that cells disrupted in either of the RPS0 genes have a reduction in growth rate and reduced amounts of 40S ribosomal subunits relative to wild-type cells. Here, we show that the Rps0 proteins are required for the processing of the 20S rRNA-precursor to mature 18S rRNA, a late step in the maturation of 40S ribosomal subunits. Immature subunits that are depleted of Rps0 protein that contain the 20S rRNA precursor are preferentially excluded from polysomes, which indicates that their activity in protein synthesis is dramatically reduced relative to mature 40S ribosomal subunits. These data demonstrate that the assembly of Rps0 proteins into immature 40S subunits and the subsequent processing of 20S rRNA represent critical steps in defining the translational capacity of yeast cells. If the function of these yeast proteins is representative of other members of the p40/37-LRP family of proteins, then the role of these proteins as key components of the protein synthetic machinery should also be considered as a basis for the linkage between the their overexpression and tumor cell growth and proliferation.     

Conformational variability of the N-terminal helix in the structure of ribosomal protein S15

The determination of the abilities of flavonoids, hydroxycinnamates and phenolics to scavenge free radicals in vitro suggests potent combined antioxidant activities of fruits, vegetables, beverages and grains. However, the key question of uptake in humans has only recently been approached consistently. The study described here demonstrates the uptake of hydroxycinnamates, for the first time, and other phenolic components, applying an HPLC method for their detection in the urine of subjects consuming levels of specific fruit equivalent to an approximate intake of 25 mg flavonol glycosides.     

Major groove binding of the tRNA/mRNA complex to the 16 S ribosomal RNA decoding site

We propose a detailed three-dimensional model, with atomic detail, for the structure of the Escherichia coli 16 S rRNA decoding site in a complex with mRNA and the A and P-site tRNAs. Model building began with four primary assumptions: (1) A and P-site tRNA conformations are identical with those seen in the tRNA crystal structure; (2) A and P-site tRNAs adopt an S-type orientation upon binding mRNA in the ribosome; (3) A1492 and A1493 bind non-specifically to the mRNA through a series of hydrogen bonds; and (4) C1400 lies in close proximity to the P-site tRNA wobble base in order to satisfy a UV-induced photocrosslink formed between the two residues. We have models with both major groove and minor groove binding of the tRNA/mRNA complex to the decoding site RNA, and conclude that major groove binding is more likely. Both classes of models maintain structural features reported in the NMR structure of the A-site region of the decoding site RNA with bound paromomycin. We also present models for the tRNA/mRNA complex bound to the decoding site RNA in the presence of the aminoglycoside paromomycin. We discuss possible mechanisms for ribosomal proof reading and antibiotic disruption of this proofreading.     

Influence of DNA binding on the degradation of oxidized histones by the 20S proteasome

The 20S proteasome is localized in the cytosol and nuclei of mammalian cells. Previous work has shown that the cytosolic 20S proteasome is largely responsible for the selective recognition and degradation of oxidatively damaged cytosolic proteins. Since nuclear proteins are also susceptible to oxidative damage (e.g., from metabolic free radical production, ionizing radiation, xenobiotics, chemotherapy) we investigated the degradation of oxidatively damaged histones, in the presence and in the absence of DNA, by the 20S proteasome. We find that both soluble histones and DNA-bound histones are susceptible to selective proteolytic degradation by the 20S proteasome following mild oxidative damage. In contrast, more severe oxidative damage actually decreases the proteolytic susceptibility of histones. Soluble H1 showed the highest basal and maximal absolute proteolytic rates. Histone fraction H4 exhibited the greatest relative increase in proteolytic susceptibility following oxidation, almost 14-fold, and this occurred at a peroxide exposure of 5 mM. At the other end of the spectrum, histone H2A exhibited a maximal proteolytic response to H2O2 of only 6-fold, and this required an H2O2 exposure of 15 mM. An oxidation of reconstituted linear DNA plasmid-histone complex makes up to 95% of the histones bound to DNA susceptible to degradation, whereas undamaged protein-DNA complexes are not substrates for the proteasome. Severe oxidation by high concentrations of H2O2 appears to decreases the proteolytic susceptibility of histones due to the formation of cross-linked histone-DNA aggregates which appear to inhibit the proteasome. We conclude that the degradation of nuclear proteins is highly selective and requires prior damage of the substrate protein, such as that caused by oxidation.