Dial tm for rescue: tmRNA engages ribosomes stalled on defective mRNAs

Ribosomes translate genetic information encoded by mRNAs into protein chains with high fidelity. Truncated mRNAs lacking a stop codon will cause the synthesis of incomplete peptide chains and stall translating ribosomes. In bacteria, a ribonucleoprotein complex composed of tmRNA, a molecule that combines the functions of tRNAs and mRNAs, and small protein B (SmpB) rescues stalled ribosomes. The SmpB-tmRNA complex binds to the stalled ribosome, allowing translation to resume at a short internal tmRNA open reading frame that encodes a protein degradation tag. The aberrant protein is released when the ribosome reaches the stop codon at the end of the tmRNA open reading frame and the fused peptide tag targets it for degradation by cellular proteases. The recently determined NMR structures of SmpB, the crystal structure of the SmpB-tmRNA complex and the cryo-EM structure of the SmpB-tmRNA-EF-Tu-ribosome complex have provided first detailed insights into the intricate mechanisms involved in ribosome rescue.     

Two yeast mutants defective in endocytosis are defective in pheromone response

We have purified biosynthetically labeled alpha-factor secreted from transformed yeast alpha cells. This alpha-factor binds specifically to a cells and is internalized by a time-, temperature-, and energy-dependent process. alpha-factor is internalized in an intact form and then rapidly degraded. Two yeast mutants defective in the accumulation of an endocytotic marker, lucifer yellow CH, in the vacuole have been isolated. end1 accumulates invaginations of the plasma membrane, and end2, an internal membrane-bound organelle. One of these mutants, end1, is defective for internalization of alpha-factor. Both of these mutants are defective in pheromone response.     

Elongating ribosomes in vivo are refractory to erythromycin

We have studied the kinetics of erythromycin inhibition of translation in growing bacteria. In order to simplify the interpretation of our data, we have used a mutant (envA), known to have an increased permeability to several antibiotics, including erythromycin. The data clearly show that an initial stage of translation is sensitive to erythromycin, but that the elongating ribosome is insensitive to the antibiotic.     

Neurospora crassa cytoplasmic ribosomes: cold-sensitive mutant defective in ribosomal ribonucleic acid synthesis.

Experiments were conducted to characterize further the biochemical defects of crib-1 (PJ30201), a coldsensitive mutant strain of Neurospora crassa with a defect in ribosome biosynthesis. The results are as follows. (i) The critical temperature for the expression of the mutant growth and ribosome phenotypes is in the range of 18 to 20 C. (ii) No preferential breakdown of 37S cytoplasmic ribosomal subunits synthesized by crib-1 at 25 C occurs after a shift to 10 C. (iii) Ribosomal subunits synthesized by crib-1 at 25 C function normally in in vivo protein synthesis at 10 C. (iv) Whereas wild type synthesizes both ribosomal subunits in a coordinate manner after either a temperature shift-down (25 to 10 C) of a shift-up (10 to 25 C), noncoordinate synthesis of ribosomal subunits owing to underproduction of 37S subunits occurs in the crib-1 strain immediately after a temperature shift-down. (v) After a shift from 10 to 25 C crib-1 exhibits a 12-h lag before the growth rate and the rate of synthesis of 37S subunits begin to increase significantly. (vi) At 10 C crib-1 synthesizes unequal amounts of 25S and 17S ribosomal ribonucleic acid (rRNA) molecules, resulting from a greatly reduced accumulation of stable 17S rRNA. The mutant phenotypes of crib-1 are proposed to be the result of a defect in rRNA processing.     

Translation stress and collided ribosomes are co-activators of cGAS

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Dormant ribosomes inBlastocladiella emersonii zoospores are arrested at elongation

Ribosomes fromBlastocladiella emersonii zoospores stimulatein vitro protein synthesis in a system using soluble factors extracted from wheat germ. Aurintricar☐ylic acid inhibits less than 40% of thein vitro protein synthetic activity of the zoospore ribosomes, indicating that messenger RNA is already complexed to the ribosomes. In addition to the mRNA complexed to the ribosomes, zoospores contain an mRNA fraction which is not bound to the ribosomes. Extraction of RNA from zoospore ribosomes and deacylation followed by reacylation with labeled amino acids demonstrated the presence of tRNA molecules specific for methionine and other amino acids on zoospore ribosomes. Transfer RNA from zoospore ribosomes contained 9.8% methionyl-tRNA compared to 2.4% methionyl-tRNA bound to ribosomes isolated from growth-phase plants. The fourfold enrichment of methionyl-tRNA on zoospore ribosomes suggests that between 12 and 25% of the zoospore ribosomes exist in arrested 80 S initiation complexes. Collectively, the data indicate that zoospore ribosomes complexed to mRNA have completed initiation and are somehow blocked at one or more of the elongation steps of protein synthesis. The data are compatible with the idea that an inhibitor is associated with the zoospore ribosomesin vivo.     

Yeast N(alpha)-terminal acetyltransferases are associated with ribosomes

N-terminal acetylation is one of the most common modifications, occurring on the vast majority of eukaryotic proteins. Saccharomyces cerevisiae contains three major NATs, designated NatA, NatB, and NatC, with each having catalytic subunits Ard1p, Nat3p, and Mak3p, respectively. Gautschi et al. (Gautschi et al. [2003] Mol Cell Biol 23: 7403) previously demonstrated with peptide crosslinking experiments that NatA is bound to ribosomes. In our studies, biochemical fractionation in linear sucrose density gradients revealed that all of the NATs are associated with mono- and polyribosome fractions. However only a minor portion of Nat3p colocalized with the polyribosomes. Disruption of the polyribosomes did not cause dissociation of the NATs from ribosomal subparticles. The NAT auxiliary subunits, Nat1p and Mdm20p, apparently are required for efficient binding of the corresponding catalytic subunits to the ribosomes. Deletions of the genes corresponding to auxiliary subunits significantly diminish the protein levels of the catalytic subunits, especially Nat3p, while deletions of the catalytic subunits produced less effect on the stability of Nat1p and Mdm20p. Also two ribosomal proteins, Rpl25p and Rpl35p, were identified in a TAP-affinity purified NatA sample. Moreover, Ard1p copurifies with Rpl35p-TAP. We suggest that these two ribosomal proteins, which are in close proximity to the ribosomal exit tunnel, may play a role in NatA attachment to the ribosome.     

The formation of a defective small subunit of the mitochondrial ribosomes in petite mutants of Saccharomyces cerevisiae

The involvement of mitochondrial protein synthesis in the assembly of the mitochondrial ribosomes was investigated by studying the extent to which the assembly process can proceed in petite mutants of Saccharomyces cerevisiae which lack mitochondrial protein synthetic activity due to the deletion of some tRNA genes and/or one of the rRNA genes on the mtDNA. Petite strains which retain the 15-S rRNA gene can synthesize this rRNA species, but do not contain any detectable amounts of the small mitochondrial ribosomal subunit. Instead, a ribonucleoparticle with a sedimentation coefficient of 30 S (instead of 37 S) was observed. This ribonucleoparticle contained all the small ribosomal subunit proteins with the exception of the var1 and three to five other proteins, which indicates that the 30-S ribonucleoparticle is related to the small mitochondrial ribosomal subunit (37 S). Reconstitution experiments using the 30-S particle and the large mitochondrial ribosomal subunit from a wild-type yeast strain indicate that the 30-S particle is not active in translating the artificial message poly(U). The large mitochondrial ribosomal subunit was present in petite strains retaining the 21-S rRNA gene. The petite 54-S subunit is biologically active in the translation of poly(U) when reconstituted with the small subunit (37 S) from a wild-type strain. The above results indicate that mitochondrial protein synthetic activity is essential for the assembly of the mature small ribosomal subunit, but not for the large subunit. Since the var1 protein is the only mitochondrial translation product known to date to be associated with the mitochondrial ribosomes, the results suggest that this protein is essential for the assembly of the mature small subunit.     

Lipopolysaccharide mutants of Rhizobium meliloti are not defective in symbiosis

Mutants of Rhizobium meliloti selected primarily for bacteriophage resistance fall into 13 groups. Mutants in the four best-characterized groups (class A, lpsB, lpsC, and class D), which map to the rhizobial chromosome, appear to affect lipopolysaccharide (LPS) as judged by the reactivity with monoclonal antibodies and behavior on sodium dodecyl sulfate-polyacrylamide gels of extracted LPS. Mutations in all 13 groups, in an otherwise wild-type genetic background, are Fix+ on alfalfa. This suggests that LPS does not play a major role in symbiosis. Mutations in lpsB, however, are Fix- in one particular genetic background, evidently because of the cumulative effect of several independent background mutations. In addition, an auxotrophic mutation evidently equivalent to Escherichia coli carAB is Fix- on alfalfa.     

Human platelets are defective in processing of cholera toxin.

Cholera toxin is unable to elevate cyclic AMP levels in intact human platelets despite being very efficacious in this respect in other mammalian cells; in the presence of 0.5 mM-isobutylmethylxanthine, we found that 3-6nM-cholera toxin over 3h at 37 degrees C elevated platelet cyclic AMP from 33 +/- 13 to 39 +/- 12pmol/mg of protein (means +/- S.D.; n = 12). We have investigated the basis for this lack of response. 125I-labelled cholera toxin bound to platelets both saturably and with high affinity (Kd congruent to 60pM; Bmax. congruent to 50fmol/mg of protein). Incubation of platelets with the putative cholera toxin receptor monosialoganglioside GM1 enhanced 125I-labelled cholera toxin binding at least 40-fold but facilitated only a minimal (less than or equal to 3-fold) elevation of platelet cyclic AMP levels. In contrast, dithiothreitol-activated cholera toxin markedly stimulated adenylate cyclase activity in platelet membranes. Platelet cytosol both enhanced stimulation of adenylate cyclase activity by activated cholera toxin (A1 subunit) and supported stimulation by the A1-A2 subunit of cholera toxin. Neither GTP nor NAD+, both necessary for response to cholera toxin, was lacking in intact platelets. However, we found that platelets were unable to cleave cholera toxin to the active A1 subunit (as assessed by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis). By contrast, murine S49 lymphoma cells were able to generate the A1 subunit with a time course that closely resembled the kinetics of toxin-mediated cyclic AMP accumulation in these cells. Thus we conclude that human platelets are defective in their ability to process surface-bound cholera toxin. These results indicate that binding of cholera toxin to surface receptors is necessary, but not sufficient, for expression of the toxin effect and the generation of the A1 subunit of the toxin may be rate-limiting for expression of cholera toxin response.     

White mutants of Chlamydomonas reinhardtii are defective in phytoene synthase

Carotenoids play an integral and essential role in photosynthesis and photoprotection in plants and algae. A collection of Chlamydomonas reinhardtii mutants lacking carotenoids was characterized for pigment and tocopherol (vitamin E) composition, growth phenotypes under different light conditions, and the molecular basis of their mutant phenotype. The carotenoid-less mutants, or "white" mutants, were also deficient in chlorophylls but had approximately twice the tocopherol content of the wild type. White mutants grew in the dark but were unable to survive in the light, even under very low light conditions on acetate-containing medium. Genetic crosses and recombination tests revealed that all individual white mutants in the collection are alleles of a single gene, lts1, and the white phenotype was closely linked to a marker located in the phytoene synthase gene. DNA sequencing of the phytoene synthase gene from each of the mutants revealed nonsense, missense, frameshift, and splice site mutations. Transformation with a wild-type copy of the phytoene synthase gene was able to complement the lts1-210 mutation. Together, these results show that all the white mutants examined in this work are affected in the phytoene synthase gene.     

Photosystem I reaction center polypeptides of spinach are synthesized on thylakoid-bound ribosomes

Purified chloroplasts were prepared from developing spinach leaves. The chloroplasts were separated into thylakoid and stroma fractions, and nucleic acids were prepared from them. Photosystem I reaction center polypeptide(s) (PS I RC) mRNA was associated with the thylakoid fraction when measured by hybridization using a probe for PS I RC polypeptide ps1A1, or when measured by translation assay. The ps1A1 polypeptide was coded for by a 5.5-kbp mRNA which others have shown also codes for PS IRC polypeptide ps1A2. This mRNA was in functional thylakoid-bound ribosomes because when thylakoids with bound ribosomes were translated in the absence of protein synthesis initiation, polypeptides that reacted with anti-PS I RC were formed. The results indicate that PS I RC polypeptides are synthesized exclusively by thylakoid-bound ribosomes.     

Ram and buck management

Careful management is necessary to ensure the reproductive success in any small ruminant farm and to maximise the productive longevity of rams and bucks. Rams and bucks are frequently overlooked outside of the breeding period, but year-round attention to nutrition, parasite control and general disease control is important in keeping them healthy and sound for breeding. Pre-mating soundness examinations are an inexpensive and relatively easy way to assess the potential ability of a ram or buck to perform during the breeding period and should be incorporated annually into the management of any flock. During the breeding period, careful thought should be given to the appropriate use of males, and the effectiveness of mating should be monitored.