Structure of a novel flavin chromophore from Avena coleoptiles, the possible ''blue light'' photoreceptor

The effect of edeine on the translation of mRNA or poly(U)-directed polyphenylalanine synthesis has been studied in an edeine-resistant mutant of Saccharomyces cerevisiae under three different experimental conditions: in the whole lysate system, in a micrococcal-nuclease-treated lysate, and in a high-salt-treated lysate. The results indicate that translation of messenger is more resistant to edeine in the whole lysate than in the depleted lysates; these observations suggest that resistance to edeine is associated with the presence of endogenous mRNA. It is shown that 40S mutant subunits have a higher affinity for polysomal RNA than 40S wild-type subunits. Since the mRNA binding is inhibited by 7-methylguanosine 5'-monophosphate, the interaction between polysomal RNA and 40S ribosomes is specific for mRNA. The data demonstrate that in each of the depleted lysates, with edeine initially present, the formation of the 80S initiation complex is inhibited. However, edeine inhibition of [3H]methionine binding to 80S ribosomes is overcome completely in the mutant extract by preincubation of this lysate with polysomal RNA. The results indicate that the mutant may carry a specific change in a messenger-binding factor or in a ribosomal protein thereby permitting an increased stability of the messenger-ribosome complex which consequently results in an increased resistance of the mutant lysate to edeine.     

Edeine inhibition and resistance in Neurospora

To obtain data on the biochemical effects of edeine in the fungus Neurospora crassa, in vivo protein synthesis, in vitro protein synthesis, as well as in vivo RNA and DNA synthesis of the wildtype and an edeine resistant mutant were measured.—Incorporation of ³H leucine into conidia of both strains, which served as a measure for in vivo protein synthesis, was inhibited by 200 g edeine/ml as follows: Wildtype approx. 40%, mutant approx. 6%.—Incorporation of ¹⁴C phenylalanine into polyphenylalanine in a cell free system with ribosomes from either the wildtype or the mutant, was inhibited between 74 and 95% by edeine at a ratio of 2 molecules edeine per ribosome.—Incorporation of ³H adenosine into conidia, serving as a measure for in vivo RNA synthesis, was inhibited in the wild-type (approx. 30% inhibition by 200 g edeine/ml). It was, however, not influenced in the ed ^r mutant. Similarly, in vivo DNA synthesis was decreased in the wildtype, but not in the mutant.—These results suggest that edeine acts at more than one site. The resistance of the mutant ed ^r -29 (ed ^r -2 locus) is tentatively interpreted as due to a block in edeine uptake.     

The effect of the antibiotic edeine on tRNA interaction with A, P and E sites of Escherichia coli 70S ribosomes

Edeine inhibits poly(U)-dependent binding of tRNAPhe to the P and A sites simultaneously, both on 30S subunits and 70S ribosomes. Hence, edeine cannot be considered as antibiotic, "complementary" to tetracycline for selective adsorption of tRNA only to the P or to the A site. Further, edeine decreases the affinity constant of tRNAPhe for the P-site by more than two orders of magnitude, no matter poly(U) is present or not. Neither edeine nor tetracycline affect interaction of deacylated tRNAPhe with the E-site of E. coli 70S ribosomes.     

Hyper-accurate ribosomes inhibit growth.

We have compared both in vivo and in vitro translation by ribosomes from wild-type bacteria with those from streptomycin-resistant (SmR), streptomycin-dependent (SmD) and streptomycin-pseudo-dependent (SmP) mutants. The three mutant bacteria translate more accurately and more slowly in the absence of streptomycin (Sm) than do wild-type bacteria. In particular, the SmP bacteria grow at roughly half the rate of the wild-type in the absence of Sm. The antibiotic stimulates both the growth rate and the translation rate of SmP bacteria by approximately 2-fold, but it simultaneously increases the nonsense suppression rate quite dramatically. Kinetic experiments in vitro show that the greater accuracy and slower translation rates of mutant ribosomes compared with wild-type ribosomes are associated with much more rigorous proofreading activities of SmR, SmD and SmP ribosomes. Sm reduces the proofreading flows of the mutant ribosomes and stimulates their elongation rates. The data suggest that these excessively accurate ribosomes are kinetically less efficient than wild-type ribosomes, and that this inhibits mutant growth rates. The stimulation of the growth of the mutants by Sm results from the enhanced translational efficiency due to the loss of proofreading, which more than offsets the loss of accuracy caused by the antibiotic.     

Multiple effects of kanamycin on translational accuracy

Summary We have studied the effects of kanamycin on the accuracy of translation in vitro by wild-type and mutant ribosomes from Escherichia coli. Kanamycin stimulates the leucine missense error of poly(U) translation by wild-type, Ram, and streptomycin-resistant ribosomes in characteristic ways; in particular, the streptomycin-resistant ribosomes are significantly less error-prone than wild-type or Ram ribosomes at all concentrations of the antibiotic. Kinetic analysis of the effects of kanamycin on the translational accuracy of wild-type ribosomes reveals a different concentration dependence for the perturbation of the initial selectivity and for the proofreading. Furthermore, the initial selectivity of streptomycin-resistant ribosomes is not affected by kanamycin; the drug enhances only the error of proofreading by this mutant ribosome. We suggest that the multiple effects of kanamycin on the errors of translation are due to separate effects at different ribosomal sites.Abbreviations N-AcPhe N-acetylphenylalanine - Km Kanamycin (used in the Figures and Tables only) - Str streptomycin (-'-) - EF elongation factor - TCA trichloroacetic acid - Ram ribosome ambiguity mutant     

Streptomycin preferentially perturbs ribosomal proofreading

Summary We have studied the influence of streptomycin (Sm) on the kinetics and accuracy of translation by wild-type as well as Ram-mutant ribosomes in an in vitro system that mimics the performance characteristics of ribosomes in bacteria. It can be shown in this system that the accuracy of translation is made up of an initial selection step and one or more proofreading steps. The data show that the antibiotic has only a small influence on the initial selectivity step of wild-type or mutant ribosomes. Streptomycin stimulates the missense rate primarily by suppressing the proofreading of the ribosomes. The kinetic effects of Sm and of Ram alteration are not additive, but seem to be overlapping if not identical.     

Mutant ribosomes can generate dominant kirromycin resistance.

Mutations in the two genes for EF-Tu in Salmonella typhimurium and Escherichia coli, tufA and tufB, can confer resistance to the antibiotic kirromycin. Kirromycin resistance is a recessive phenotype expressed when both tuf genes are mutant. We describe a new kirromycin-resistant phenotype dominant to the effect of wild-type EF-Tu. Strains carrying a single kirromycin-resistant tuf mutation and an error-restrictive, streptomycin-resistant rpsL mutation are resistant to high levels of kirromycin, even when the other tuf gene is wild type. This phenotype is dependent on error-restrictive mutations and is not expressed with nonrestrictive streptomycin-resistant mutations. Kirromycin resistance is also expressed at a low level in the absence of any mutant EF-Tu. These novel phenotypes exist as a result of differences in the interactions of mutant and wild-type EF-Tu with the mutant ribosomes. The restrictive ribosomes have a relatively poor interaction with wild-type EF-Tu and are thus more easily saturated with mutant kirromycin-resistant EF-Tu. In addition, the mutant ribosomes are inherently kirromycin resistant and support a significantly faster EF-Tu cycle time in the presence of the antibiotic than do wild-type ribosomes. A second phenotype associated with combinations of rpsL and error-prone tuf mutations is a reduction in the level of resistance to streptomycin.     

The contribution of the zinc-finger motif to the function of Thermus thermophilus ribosomal protein S14

In the crystal structure of the 30S ribosomal subunit from Thermus thermophilus, cysteine 24 of ribosomal protein S14 (TthS14) occupies the first position in a CXXC-X12-CXXC motif that coordinates a zinc ion. The structural and functional importance of cysteine 24, which is widely conserved from bacteria to humans, was studied by its replacement with serine and by incorporating the resulting mutant into Escherichia coli ribosomes. The capability of such modified ribosomes in binding tRNA at the P and A-sites was equal to that obtained with ribosomes incorporating wild-type TthS14. In fact, both chimeric ribosomal species exhibited 20% lower tRNA affinity compared with native E. coli ribosomes. In addition, replacement of the native E. coli S14 by wild-type, and particularly by mutant TthS14, resulted in reduced capability of the 30S subunit for association with 50S subunits. Nevertheless, ribosomes from transformed cells sedimented normally and had a full complement of proteins. Unexpectedly, the peptidyl transferase activity in the chimeric ribosomes bearing mutant TthS14 was much lower than that measured in ribosomes incorporating wild-type TthS14. The catalytic center of the ribosome is located within the 50S subunit and, therefore, it is unlikely to be directly affected by changes in the structure of S14. More probably, the perturbing effects of S14 mutation on the catalytic center seem to be propagated by adjacent intersubunit bridges or the P-site tRNA molecule, resulting in weak donor-substrate reactivity. This hypothesis was verified by molecular dynamics simulation analysis.     

Characterization of a cycloheximide-resistant Tetrahymena thermophila mutant which also displays altered growth properties.

A cycloheximide-resistant strain of Tetrahymena thermophila, expressing a mutant chx-B gene (Ares and Bruns, Genetics 90:463-474, 1978), displayed very different temperature-dependent growth characteristics than either wild-type cells or another cycloheximide-resistant strain expressing a different mutant gene. Whereas wild-type cells showed an immediate decline in ribosome translocation rates when shifted from 30 to 38 or 40 degrees C, this mutant strain (X-8) showed no such decline. These results directly correlated with the growth rate differences we found for these cells at these temperatures. By genetic analysis, we showed that the phenotype of cycloheximide resistance cosegregated with the ability to grow rapidly at 40 degrees C. Analyses, both direct and indirect, suggested that a number of functional and structural characteristics of the ribosomes from strain X-8 cells are most likely conformationally different from those of wild-type ribosomes.     

Altered chloroplast ribosomal proteins in a yellow mutant of Chlamydomonas reinhardii.

Summary Ribosomes and ribosomal proteins from wild-type and a yellow mutant of Chlamydomonas reinhardii were analysed and compared by two-dimensional gel electrophoresis.Mixothrophycally grown yellow-27 mutant differs from wild-type cells in lowered chlorophyll content and grana fromation of the chloroplast.Analytical ultracentrifuge analyses of cell extracts show a reduced amount of free 70S ribosomes and increased level of 50S subunits in the mutant cells. Similar results were obtained by electronmicroscopical method.Two-dimensional gel electrophoresis shows alterations in protein composition of 70S ribosomes of the mutant. Two proteins of 70S ribosomes have been altered. One of them with high molecular weight is practically absent while there is an additional, intensively stained spot in the mutant.Since the mutation is inherited in a non-Mendelian manner it is possible that the protein alterations in 70S ribosome are localized in the chloroplast DNA.     

Migration of 40 S ribosomal subunits on messenger RNA when initiation is perturbed by lowering magnesium or adding drugs.

Migration of 40 S ribosomal subunits on messenger RNA, detected previously in experiments using the antibiotic edeine (Kozak, M., and Shatkin, A.J. (1978) J. Biol. Chem. 253, 6568-6577) has now been observed in the presence of other inhibitors of initiation. 40 S subunit migration has been detected in both wheat germ and reticulocyte lysates treated with edeine, pactamycin, or sodium fluoride. The variety of structurally unrelated inhibitors that mediate this effect argues against the interpretation that migration is a drug-induced artifact. Indeed, limited migration of 40 S ribosomes occurs upon simply lowering the magnesium concentration, in the absence of inhibitors. Thus, migration seems to be an inherent property of 40 S ribosomal subunits and might be involved in the mechanism by which eukaryotic ribosomes select initiation sites in messenger RNA.     

Ribosomal RNA and protein mutants resistant to spectinomycin.

We have compared the influence of spectinomycin (Spc) on individual partial reactions during the elongation phase of translation in vitro by wild-type and mutant ribosomes. The data show that the antibiotic specifically inhibits the elongation factor G (EF-G) cycle supported by wild-type ribosomes. In addition, we have reproduced the in vivo Spc resistant phenotype of relevant ribosome mutants in our in vitro translation system. In particular, three mutants with alterations at position 1192 in 16S rRNA as well as an rpsE mutant with an alteration of protein S5 were analysed. All of these ribosomal mutants confer a degree of Spc resistance for the EF-G cycle in vitro that is correlated with the degree of growth rate resistance to the antibiotic in culture.     

The binding sites for tRNA on eukaryotic ribosomes.

We have studied the non-enzymic binding of phe-tRNA to ribosomes from rat liver using deacylated tRNA to inhibit binding to the P-site and puromycin (5 x 10-minus3M) to inhibit binding to the A-site. We conclude that at a low concentration of magnesium ions (10mM) phe-tRNA is bound only at the A-site of 80S irbosomes, whereas at a high concentration of magnesium ions (40mM) phe-tRNA is also bound at the P-site. Studies with edeine indicate that, during non-enzymic binding of phe-tRNA, eukaryotic ribosomes (in contrast to prokarotic ribosomes) have the A-site of the 60S subunit and the initiation site of the 40S subunit juxtaposed. This may account for the differences observed, in formation of diphenylalanyl-tRNA and phenylalanyl-puromycin, between phe-tRNA bound non-enzymically to the P-sites of eukaryotic and prokaryotic ribosomes.     

Replacement of the L11 binding region within E.coli 23S ribosomal RNA with its homologue from yeast: in vivo and in vitro analysis of hybrid ribosomes altered in the GTPase centre.

Replacement of the protein L11 binding domain within Escherichia coli 23S ribosomal RNA (rRNA) by the equivalent region from yeast 26S rRNA appeared to have no effect on the growth rate of E.coli cells harbouring a plasmid carrying the mutated rrnB operon. The hybrid rRNA was correctly processed and assembled into ribosomes, which accumulated normally in polyribosomes. Of the total ribosomal population, < 25% contained wild-type, chromosomally encoded rRNA; the remainder were mutant. The hybrid ribosomes supported GTP hydrolysis dependent upon E.coli elongation factor G, although at a somewhat reduced rate compared with wild-type particles, and were sensitive to the antibiotic, thiostrepton, a potent inhibitor of ribosomal GTPase activity that binds to 23S rRNA within the L11 binding domain. That thiostrepton could indeed bind to the mutant ribosomes, although at a reduced level relative to that seen with wild-type ribosomes, was confirmed in a non-equilibrium assay. The rationale for the ability of the hybrid ribosomes to bind the antibiotic, given that yeast ribosomes do not, was provided when yeast rRNA was shown by equilibrium dialysis to bind thiostrepton only 10-fold less tightly than did E.coli rRNA. The extreme conservation of secondary, but not primary, structure in this region between E.coli and yeast rRNAs allows the hybrid ribosomes to function competently in protein synthesis and also preserves the interaction with thiostrepton.     

Interaction of the cytoplasmic membrane and ribosomes in Escherichia coli; altered ribosomal proteins in sucrose-dependent spectinomycin-resistant mutants.

Alterations in the ribosomes of sucrose-dependent spectinomycin-resistant (Sucd-Spcr) mutants of Escherichia coli were studied. Subunit exchange experiments showed that 30S subunits were responsible for the resistance of ribosomes to spectinomycin in all Sucd-Spcr mutants tested. Proteins of 30S ribosomes were analyzed by carboxymethyl cellulose column chromatography based on their elution positions. Mutants YM22 and YM93 had an altered 30S ribosomal protein component, S5, and mutant YM50 had an altered protein, S4. Although a shift of elution position was not detected for all the 30S ribosomal proteins from mutant YM101, the amount of protein S3 was appreciably lowered in the isolated 30S subunits. A partial reconstitution experiment with protein S3 prepared from both the wild-type strain and YM101 revealed that the mutant had altered protein S3 which is responsible for the spectinomycin resistance. These alterations in 30S subunits are discussed in relation to the interaction between ribosomes and the cytoplasmic membrane.     

Stability of protein synthesis initiation complexes in the presence of edeine

The antibiotic inhibitor edeine has been used to study various aspects of the initiation of protein synthesis in a rabbit reticulocyte cell-free system. Edeine prevents assembly of the 80S initiation complex while allowing accumulation of a 44S initiation intermediate. The complete 80S initiation complex, once formed, is stable in the presence of edeine. The functioning of the initiation complex, as judged by release of methionyl-puromycin, is only partially inhibited by a concentration of edeine which fully inhibits formation of the initiation complex. The above effects of edeine on a eukaryotic system differ from the effects edeine has been found to have in a prokaryotic system.     

Chloroplast and Cytoplasmic Ribosomes of Euglena: Selective Binding of Dihydrostreptomycin to Chloroplast Ribosomes

Dihydrostreptomycin binds preferentially to chloroplast ribosomes of wild-type Euglena gracilis Klebs var. bacillaris Pringsheim. The K(diss) for the wild-type chloroplast ribosome-dihydrostreptomycin complex is 2 x 10(-7) M, a value comparable with that found for the Escherichia coli ribosome-dihydrostreptomycin complex. Chloroplast ribosomes isolated from the streptomycin-resistant mutant Sm(1) (r)BNgL and cytoplasmic ribosomes from wild-type have a much lower affinity for the antibiotic. The K(diss) for the chloroplast ribosome-dihydrostreptomycin complex of Sm(1) (r) is 387 x 10(-7) M, and the value for the cytoplasmic ribosome-dihydrostreptomycin complex of the wild type is 1,400 x 10(-7) M. Streptomycin competes with dihydrostreptomycin for the chloroplast ribosome binding site, and preincubation of streptomycin with hydroxylamine prevents the binding of streptomycin to the chloroplast ribosome. These results indicate that the inhibition of chloroplast development and replication in Euglena by streptomycin and dihydrostreptomycin is related to the specific inhibition of protein synthesis on the chloroplast ribosomes of Euglena.     

Structural changes in the 530 loop of Escherichia coli 16S rRNA in mutants with impaired translational fidelity.

The higher order structure of the functionally important 530 loop in Escherichia coli 16S rRNA was studied in mutants with single base changes at position 517, which significantly impair translational fidelity. The 530 loop has been proposed to interact with the EF-Tu-GTP-aatRNA ternary complex during decoding. The reactivity at G530, U531 and A532 to the chemical probes kethoxal, CMCT and DMS respectively was increased in the mutant 16S rRNA compared with the wild-type, suggesting a more open 530 loop structure in the mutant ribosomes. This was supported by oligonucleotide binding experiments in which probes complementary to positions 520-526 and 527-533, but not control probes, showed increased binding to the 517C mutant 70S ribosomes compared with the non-mutant control. Furthermore, enzymatic digestion of 70S ribosomes with RNase T1, specific for single-stranded RNA, substantially cleaved both wild-type and mutant rRNAs between G524 and C525, two of the nucleotides involved in the 530 loop pseudoknot. This site was also cleaved in the 517C mutant, but not wild-type rRNA, by RNase V1. Such a result is still consistent with a more open 530 loop structure in the mutant ribosomes, since RNase V1 can cut at appropriately stacked single-stranded regions of RNA. Together these data indicate that the 517C mutant rRNA has a rather extensively unfolded 530 loop structure. Less extensive structural changes were found in mutants 517A and 517U, which caused less misreading. A correlation between the structural changes in the 530 loop and impaired translational accuracy is proposed.     

Mutations in ribosomal proteins S4 and S12 influence the higher order structure of 16 S ribosomal RNA

We have studied the effects of protein mutations on the higher order structure of 16 S rRNA in Escherichia coli ribosomes, using a set of structure-sensitive chemical probes. Ten mutant strains were studied, which contained alterations in ribosomal proteins S4 and S12, including double mutants containing both altered S4 and S12. Two ribosomal ambiguity (ram) S4 mutant strains, four streptomycin resistant (SmR) S12 mutant strains, one streptomycin pseudodependent (SmP) S12 mutant strain, one streptomycin dependent (SmD) S12 mutant strain and two streptomycin independent (Sm1) double mutants (containing both-SmD and ram mutations) were probed and compared to an isogenic wild-type strain. In ribosomes from strains containing S4 ram mutations, nucleotides A8 and A26 become more reactive to dimethyl sulfate (DMS) at their N-1 positions. In ribosomes from strains bearing the SmD allele, A908, A909, A1413 and G1487 are significantly less reactive to chemical probes. These same effects are observed when the S4 and S12 mutations are present simultaneously in the double mutants. An interesting correlation is found between the reactivity of A908 and the miscoding potential of SmR, SmD, SmP and wild-type ribosomes; the reactivity of A908 increases as the translational error frequency of the ribosomes increases. In the case of ram ribosomes, the reactivity of A908 resembles that of wild-type, unless tRNA is bound, in which case it becomes hyper-reactive. Similarly, streptomycin has little effect on A908 in wild-type ribosomes unless tRNA is bound, in which case its reactivity increases to resemble that of ram ribosomes with bound tRNA. Finally, interaction of streptomycin with SmP and SmD ribosomes causes the reactivity of A908 to increase to near-wild-type levels. A simple model is proposed, in which the reactivity of A908 reflects the position of an equilibrium between two conformational states of the 30 S subunit, one of which is DMS-reactive, and the other DMS-unreactive. In this model, the balance between these two states would be influenced by proteins S4 and S12. Mutations in S12 generally cause a shift toward the unreactive conformer, and in the case of SmD and SmP ribosomes, this shift can be suppressed phenotypically by streptomycin, ram mutations in protein S4 cause a shift toward the reactive conformer, but only when tRNA is bound. This suggests that the opposing effects of these two classes of mutations influence the proof-reading process by somewhat different mechanisms.     

An unusual precursor of 50S ribosomes in a mutant of Escherichia coli.

Escherichia coli strain 15-28 is a mutant with a defect in ribosome synthesis that leads to the accumulation of large amounts of ribonucleoprotein ("47S") particles during exponential growth. These particles are precursors to 50S ribosomes, but are distinct from precursors detected by pulse-labelling of the parent strain and also from ribosome precursors that accumulate during inhibition of growth by CoC12. Either ribosome assembly in the mutant differs from that in the wild-type strain, or 47S particles represent a hitherto unstudied stage in the synthesis of 50S ribosomes.