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1.
R. Storck 《Biophysical journal》1963,3(1):1-10
Ribosomes isolated from growing hyphae of Neurospora crassa contain 53 per cent protein and 47 per cent RNA and have a sedimentation coefficient of 81S at 20°C and infinite dilution. These ribosomes are stable at pH 7.4 in the presence of 0.01 M and 0.002 M MgCl2 but undergo a dissociation into smaller particles if the MgCl2 concentration is lowered to 0.0001 M. Two types of RNA with sedimentation coefficients of 19S2050 and 13S2050 have been extracted from the 81S particles. 相似文献
2.
Abstract A method for preparing ribosomes from Neurospora crassa is described in which che ribosomes prepared are free from the enzymes cytochrome c oxidase and NADPH-cytochrome c reductase, DNA and phospholipid material. 相似文献
3.
M. Duane Smith Yu Gu Jordi Querol-Audí Jacob M. Vogan Adam Nitido Jamie H. D. Cate 《PloS one》2013,8(11)
Eukaryotic translation initiation factor 3 (eIF3) is a key regulator of translation initiation, but its in vivo assembly and molecular functions remain unclear. Here we show that eIF3 from Neurospora crassa is structurally and compositionally similar to human eIF3. N. crassa eIF3 forms a stable 12-subunit complex linked genetically and biochemically to the 13th subunit, eIF3j, which in humans modulates mRNA start codon selection. Based on N. crassa genetic analysis, most subunits in eIF3 are essential. Subunits that can be deleted (e, h, k and l) map to the right side of the eIF3 complex, suggesting that they may coordinately regulate eIF3 function. Consistent with this model, subunits eIF3k and eIF3l are incorporated into the eIF3 complex as a pair, and their insertion depends on the presence of subunit eIF3h, a key regulator of vertebrate development. Comparisons to other eIF3 complexes suggest that eIF3 assembles around an eIF3a and eIF3c dimer, which may explain the coordinated regulation of human eIF3 levels. Taken together, these results show that Neurospora crassa eIF3 provides a tractable system for probing the structure and function of human-like eIF3 in the context of living cells. 相似文献
4.
《The International journal of biochemistry》1994,26(12):1391-1393
A computer comparison of protein sequences revealed similarity between the 30.4 kDa subunit of complex I from the fungus Neurospora crassa and the ORF5 subunit of formate hydrogenlyase from Escherichia coli. The ORF5 protein was previously known to be homologous to the 49 kDa component of the mitochondrial enzyme. We show that the 30.4 kDa corresponds to the N-terminal part while the 49 kDa subunit corresponds to the C-terminal portion of the bacterial protein. Thus, this bacterial protein represents a fusion of the two mitochondrial polypeptides suggesting that the two complex I genes arose from a single ancestor. Our results indicate that the 30.4 kDa and 49 kDa subunits are part of a structural and functional unit in complex I. 相似文献
5.
Bruno Pereira Arnaldo Videira Margarida Duarte 《Molecular and cellular biology》2013,33(13):2623-2634
Complex I deficiency is commonly associated with mitochondrial oxidative phosphorylation diseases. Mutations in nuclear genes encoding structural subunits or assembly factors of complex I have been increasingly identified as the cause of the diseases. One such factor, NDUFAF2, is a paralog of the NDUFA12 structural subunit of the enzyme, but the mechanism by which it exerts its function remains unknown. Herein, we demonstrate that the Neurospora crassa NDUFAF2 homologue, the 13.4L protein, is a late assembly factor that associates with complex I assembly intermediates containing the membrane arm and the connecting part but lacking the N module of the enzyme. Furthermore, we provide evidence that dissociation of the assembly factor is dependent on the incorporation of the putative regulatory module composed of the subunits of 13.4 (NDUFA12), 18.4 (NDUFS6), and 21 (NDUFS4) kDa. Our results demonstrate that the 13.4L protein is a complex I assembly factor functionally conserved from fungi to mammals. 相似文献
6.
7.
During trans-translation, stalled bacterial ribosomes are rescued by small protein B (SmpB) and by transfer-messenger RNA (tmRNA). Stalled ribosomes switch translation from the defective messages to a short internal reading frame on tmRNA that tags the nascent peptide chain for degradation and recycles the ribosomes. We present evidences that SmpB binds the large and small ribosomal subunits in vivo and in vitro. The binding between SmpB and the ribosomal subunits is very tight, with a dissociation constant of 1.7 × 10−10 M, similar to its KD for the 70S ribosome or for tmRNA. tmRNA displaces SmpB from its 50S binding but not from the 30S. In vivo, SmpB is detected on the 50S when trans-translation is impaired by lacking tmRNA or a functional SmpB. SmpB contacts the large subunit transiently and early during the trans-translational process. The affinity of SmpB for the two ribosomal subunits is modulated by tmRNA in the course of trans-translation. It is the first example of two copies of the same protein interacting with two different functional sites of the ribosomes. 相似文献
8.
Ribosomes from rice embryos (Oryza sativa) were dissociated into ribosomal subunits in vitro by systematic reduction of the Mg2+ concentration. Ribosomes from imbibed (28 C) embryos were more easily dissociated than those from nonimbibed embryos. This was not observed with ribosomes from either imbibed, nonviable embryos, or from embryos imbibed at 0 C. Ribosomes from embryos which had been imbided and subsequently dehydrated resembled ribosomes from nonimbibed embryos in their resistance to dissociation. The change in the resistance to dissociation was essentially complete after the first 20 minutes of imbibition at 28 C, and accompanied activation in vivo of protein synthesis as determined by amino acid incorporation in vitro. Ribosomes from either imbibed or nonimbibed embryos could be dissociated into subunits by 0.5 m KCl. These subunits were separated by density gradient centrifugation, and, if recombined, were active for polyphenylalanine synthesis in vitro. The individual subunits prepared from nonimbibed embryos could be replaced by the corresponding subunit fraction from imbibed embryos without loss of capacity to support polyphenylalanine synthesis. The change in the ease of dissociation of ribosomes appears to be a physiological process, and its possible relationship to the initiation of protein synthesis during seed germination is discussed. 相似文献
9.
These studies were designated to investigate the effect of chlortetracycline on sedimentation properties of polysomes and
ribosomes present in the chlortetracycline producing strain ofStreptomyces aureofaciens. In presence of chlortetracycline polysomes and ribosomes are more stable than the bacterial ones. At lower chlortetracycline
concentrations (1–5 μg/ml) dissociation of polysomes into 70 S monomers was not observed. Ribosomes in higher concentration
of chlortetracycline (400 μg/ml) form aggregates. A decrease of Mg2+ to 0.1mm caused dissociation of ribosomes to two subunits and in this state none of indicated concentrations of chlortetracycline
caused aggregation. The exact sedimentation values of ribosomes and ribosomal subunits were calculated from extrapolation
to infinite dilution. S20,w for monomer form was 68.8, and for ribosomal subunits 49.8 and 31.2 respectively. Ribosomal RNA sedimentates as two Schlieren
peaks of 16 S and 22 S. It was found that 30 S subunits contain 15 structural proteins, while 21 proteins were resolved from
50 S subunits. 相似文献
10.
《Experimental mycology》1980,4(1):17-22
The purpose of this investigation was to use tritium-suicide enrichment with a mutagenized population of wild-typeNeurospora crassa to isolate cold-sensitive mutants with conditional defects in the production of cytoplasmic ribosomes. Eighty-six cold-sensitive mutant strains were obtained following tritium-suicide enrichment using [5-3H]uridine. Zone sedimentation analysis of cytoplasmic ribosomes produced by the strains at 10°C (the nonpermissive temperature) indicated that one strain,PJ31562, is defective in the accumulation of ribosomal subunits at that temperature. The properties of strainPJ31562 are: (1) At 10°C the growth rate is 28 times slower than at 25°C, whereas the factor for the wild type is 5.1. At 25°C the mutant's growth rate is 90% that of the wild type. (2) At 10°C the mutant accumulates the two ribosomal subunits, 60 and 37 S, in markedly disproportionate amounts apparently as a result of the underproduction of, or an instability of, the 17 S ribosomal RNA component of the small ribosomal subunit. At 25°C the mutant strain still exhibits a disproportionality in ribosomal subunit accumulation but to a much lesser degree than at 10°C. (3) Genetic studies have shown that a single nuclear gene is responsible for both the cold sensitivity and ribosome biosynthesis defect of strainPJ31562. The mutation involved is located in linkage group IV and appears to be closely linked to, and not allelic with, the cold-sensitive mutation carried by strainPJ30201 which has been shown previously to exhibit a similar phenotype with respect to ribosomal subunit accumulation, and which defines thecrib-1 locus. Thus tritium-suicide enrichment can be used to isolate cold-sensitive mutants ofNeurospora among which a relatively low frequency have conditional defects in ribosome production. 相似文献
11.
A rapid and sensitive assay has been developed for the factor-dependent dissociation of eukaryotic ribosomes. This assay takes advantage of the observation that initiation factor eIF-2 will bind Met-tRNAfmet to 40 S subunits but not to 80 S ribosomes. Incubation of wheat germ ribosomes at 1 mm Mg2+ results in their dissociation into 40 S subunits. These subunits spontaneously reassociate when the Mg2+ concentration is raised to 4 mm. However, if the incubation at 1 mm Mg2+ is carried out in the presence of an extract containing a ribosome dissociation factor, a certain portion of the subunits will fail to reassociate when the Mg2+ concentration is raised to 4 mm. The 40 S subunits remaining due to the presence of the dissociation factor can bind [35S]Met-tRNAfmet in the presence of wheat germ eIF-2. The [35S]Met-tRNAfmet bound to the 40 S subunits is readily detected by its retention on a Millipore filter. 相似文献
12.
Cytoplasmic ribosomes were isolated and purified from sonicates of the mycelial and yeastlike growth forms of the pathogenic dimorphic fungi, Histoplasma capsulatum and Blastomyces dermatitidis. Similar ribosomal fractions were prepared from Neurospora crassa and Saccharomyces cerevisiae. These latter organisms were selected as typical filamentous and yeastlike monophasic fungi, and their ribosomes were used as reference standards. High resolution electron microscopy permitted a comparison of both positively and negatively-stained ribosomes to those dehydrated without heavy metal salt. Such studies revealed statistically significant differences in physical dimensions. Cautious interpretations of substructural detail of the various ribosomal preparations suggested both interphasic and interspecies differences. 相似文献
13.
Structural dynamics of bacterial ribosomes. I. Characterization of vacant couples and their relation to complexed ribosomes 总被引:28,自引:0,他引:28
Ribosomes not engaged in protein synthesis (vacant couples), in contrast to complexed ribosomes bearing nascent chains, dissociate during sedimentation in sucrose gradients at high g forces and at Mg2+ concentrations below 15 mm. As a result of this dissociation, a new peak between the 70 S complexed ribosomes and the free 50 S subunits is observed, the position of which shifts from about 55 S to 70 S as the Mg2+ concentration in the gradient is raised from 5 to 15 mm. The apparent 60 S peak consists of 50 S subunits produced during dissociation in the gradient. At low g forces, the sedimentation rate of complexed and vacant ribosomes is indistinguishable, even at 5 mm-Mg2+. These sedimentation properties are valid criteria to differentiate vacant and complexed ribosomes. This is shown by converting complexed ribosomes quantitatively into vacant couples by removing the nascent chains through termination release or with puromycin, or by converting vacant couples into initiation complexes with R17 RNA, fMet-tRNA and initiation factors.Ribosomes from cells harvested by slow cooling consist almost entirely of vacant couples, all of which are active in protein synthesis with natural messengers. The structural features responsible for the interaction between subunits are discussed. 相似文献
14.
Joseph Keesey James Paukert John A. Demoss 《Archives of biochemistry and biophysics》1981,207(1):103-109
The multifunctional enzyme complex anthranilate synthase from Neurospora crassa has been purified to homogeneity by a new procedure which yields a stable preparation of the enzyme. Unlike earlier preparations of the enzyme, anthranilate synthase prepared by this technique is not degraded during incubation at 37 °C or during freeze-thaw treatment. Purified anthranilate synthase contains two subunits of Mr 84,000 (β-subunit) and 76,000 (α-subunit), which are shown, by partial proteolysis, to be unrelated in sequence. Immunoprecipitation studies demonstrate that freshly prepared crude extracts of Neurospora contain anthranilate synthase subunits identical in size with those of the purified enzyme. The β-subunit is shown to be the product of the trp1 gene, and the a-subunit, of the trp2 gene. 相似文献
15.
Summary In Neurospora crassa, mutants resistant against cycloheximide appear with a marked time lag after mutation induction. We have suggested (Neuhäuser et al., 1970) that this lag indicates the time needed for the synthesis of altered ribosomes (phenotypic lag), that the drug in the wildtype acts upon the ribosomes, and that resistance is due to alterations in them.By measurements of poly-U directed poly-Phe synthesis on ribosomes of the wildtype and two different cycloheximide resistant mutants in a cell free system it is shown here that mutant ribosomes indeed differ from those of the wildtype. Poly-Phe synthesis on mutant ribosomes proceeds in the presence of the drug, whereas that on wildtype ribosomes is inhibited. This means that the earlier suggestions are correct.Abbreviation CHX
cycloheximide 相似文献
16.
Abstract Ribosomes prepared from the bacterium Bacillus subtilis, from mitochondria of Neurospora crassa and from chloroplasts of Euglena gracilis carry out the ApUpG-dependent reactions for peptide chain initiation demonstrated for Escherichia coli ribosomes. Initiation factors functionally similar to those from E. coli appear to be present on the above reported ribosomal preparations. The activity of ribosomes washed with high concentrations of ammonium chloride may be restored by adding initiation factors prepared from E. coli. 相似文献
17.
Karen S. Stephenson Neil A.R. Gow Fordyce A. Davidson Geoffrey M. Gadd 《Fungal biology》2014,118(3):287-294
Thigmotropism is the ability of an organism to respond to a topographical stimulus by altering its axis of growth. The thigmotropic response of the model fungus Neurospora crassa was quantified using microfabricated glass slides with ridges of defined height. We show that the polarity machinery at the hyphal tip plays a role in the thigmotropic response of N. crassa. Deletion of N. crassa genes encoding the formin, BNI-1, and the Rho-GTPase, CDC-42, an activator of BNI-1 in yeast, CDC-24, its guanine nucleotide exchange factor (GEF), and BEM-1, a scaffold protein in the same pathway, were all shown to significantly decrease the thigmotropic response. In contrast, deletion of genes encoding the cell end-marker protein, TEA-1, and KIP-1, the kinesin responsible for the localisation of TEA-1, significantly increased the thigmotropic response. These results suggest a mechanism of thigmotropism involving vesicle delivery to the hyphal tip via the actin cytoskeleton and microtubules. Neurospora crassa thigmotropic response differed subtly from that of Candida albicans where the stretch-activated calcium channel, Mid1, has been linked with thigmotropic behaviour. The MID-1 deficient mutant of N. crassa (Δmid-1) and the effects of calcium depletion were examined here but no change in the thigmotropic response was observed. However, SPRAY, a putative calcium channel protein, was shown to be required for N. crassa thigmotropism. We propose that the thigmotropic response is a result of changes in the polarity machinery at the hyphal tip which are thought to be downstream effects of calcium signalling pathways triggered by mechanical stress at the tip. 相似文献
18.
Release of 70 S ribosomes from polysomes in Escherichia coli 总被引:5,自引:0,他引:5
In order to determine whether ribosomes are released from messenger RNA as intact particles or as subunits, polysomes of Escherichia coli labeled with heavy isotopes were allowed to run off together with “light” polysomes. The normally rapid post-run-off exchange of subunits by free ribosomes was virtually eliminated by two means: the use of purified polysomes (relatively free of initiation factors), and incubation at a lower temperature (25 °C), or at a somewhat higher Mg2+ concentration (12 to 14 mm), than is conventional. Under these conditions ribosomes released by run-off or by puromycin accumulated without subunit exchange. Hence, even though the ribosome normally initiates via subunits, it is released from RNA by a conformational change in the intact 70 S particle, rather than by dissociation. 相似文献
19.
Purified eukaryotic initiation factor, EIF-3, prepared from ascites cells dissociated rat liver 80S ribosomes into subunits. Ribosomes bearing endogenous mRNA and nascent peptide were not dissociated by EIF-3. When 80S ribosomes reconstituted from subunits were used as substrate the reaction had the following characteristics: Dissociation was rapid--the reaction being completed within 2 min at 30°. The extent of dissociation was directly proportional to the amount of EIF-3; with 21 μg of EIF-3 about 70% (or 10.5 μg) of the 80S monomers were dissociated. The dissociation of 80S monomers by EIF-3 decreased with increasing concentrations of magnesium. The reaction was not catalytic: 28 moles of EIF-3 were required to dissociate 1 mole of 80S ribosomes. The characteristic of the dissociation reaction promoted by EIF-3 and by initiation factor IF-3 are remarkably similar. The dissociation reaction provides a practical assay for EIF-3 independent of complimentation of other initiating factors. 相似文献
20.
THE INTRACELLULAR SITE OF SYNTHESIS OF MITOCHONDRIAL RIBOSOMAL PROTEINS IN NEUROSPORA CRASSA 总被引:8,自引:3,他引:5
The intracellular site of synthesis of mitochondrial ribosomal proteins (MRP) in Neurospora crassa has been investigated using three complementary approaches. (a) Mitochondrial protein synthesis in vitro: Tritium-labeled proteins made by isolated mitochondria were compared to 14C-labeled marker MRP by cofractionation in a two-step procedure involving isoelectric focusing and polyacrylamide gel electrophoresis. Examination of the electrophoretic profiles showed that essentially none of the peaks of in vitro product corresponded exactly to any of the MRP marker peaks. (b) Sensitivity of in vivo MRP synthesis to chloramphenicol: Cells were labeled with leucine-3H in the presence of chloramphenicol, mitochondrial ribosomal subunits were subsequently isolated, and their proteins fractionated by isoelectric focusing followed by gel electrophoresis. The labeling of every single MRP was found to be insensitive to chloramphenicol, a selective inhibitor of mitochondrial protein synthesis. (c) Sensitivity of in vivo MRP synthesis to anisomycin: We have found this antibiotic to be a good selective inhibitor of cytoplasmic protein synthesis in Neurospora. In the presence of anisomycin the labeling of virtually all MRP is inhibited to the same extent as the labeling of cytoplasmic ribosomal proteins. On the basis of these three types of studies we conclude that most if not all 53 structural proteins of mitochondrial ribosomal subunits in Neurospora are synthesized by cytoplasmic ribosomes. 相似文献