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1.
A complex of acidic ribosomal proteins. Evidence of a four-to-one complex of proteins in the Bacillus stearothermophilus ribosome 总被引:2,自引:0,他引:2
Two acidic proteins from the 50 S subunit of Bacillus stearothermophilus ribosomes, namely B-L13 (homologous to Escherichia coli protein ) and B-L8, form a complex. Radioactive B-L13, added to ribosomes before dissociation, does not appear in the complex after electrophoresis, so the (B-L13 · B-L8) complex must exist in the ribosome before dissociation. Digestion of B. stearothermophilus ribosomes with polyacrylamide-bound trypsin causes the appearance of new B-L8 and B-L13 spots on two-dimensional polyacrylamide gel electrophoresis, in a pattern which suggests that single molecules of B-L13 are being sequentially cleaved from a four-to-one complex of B-L13 and B-L8. 相似文献
2.
Effects of site-directed mutations in the central domain of 16 S ribosomal RNA upon ribosomal protein binding, RNA processing and 30 S subunit assembly 总被引:10,自引:0,他引:10
M J Stark R J Gregory R L Gourse D L Thurlow C Zwieb R A Zimmermann A E Dahlberg 《Journal of molecular biology》1984,178(2):303-322
3.
Interaction of ribosomal proteins S6, S8, S15 and S18 with the central domain of 16 S ribosomal RNA from Escherichia coli 总被引:12,自引:0,他引:12
R J Gregory M L Zeller D L Thurlow R L Gourse M J Stark A E Dahlberg R A Zimmermann 《Journal of molecular biology》1984,178(2):287-302
The co-operative interaction of 30 S ribosomal subunit proteins S6, S8, S15 and S18 with 16 S ribosomal RNA from Escherichia coli was studied by (1) determining how the binding of each protein is influenced by the others and (2) characterizing a series of protein-rRNA fragment complexes. Whereas S8 and S15 are known to associate independently with the 16 S rRNA, binding of S18 depended upon S8 and S15, and binding of S6 was found to require S8, S15 and S18. Ribonucleoprotein (RNP) fragments were derived from the S8-, S8/S15- and S6/S8/S15/S18-16 S rRNA complexes by partial RNase hydrolysis and isolated by electrophoresis through Mg2+-containing polyacrylamide gels or by centrifugation through sucrose gradients. Identification of the proteins associated with each RNP by gel electrophoresis in the presence of sodium dodecyl sulfate demonstrated the presence of S8, S8 + S15 and S6 + S8 + S15 + S18 in the corresponding fragment complexes. Analysis of the rRNA components of the RNP particles confirmed that S8 was bound to nucleotides 583 to 605 and 624 to 653, and that S8 and S15 were associated with nucleotides 583 to 605, 624 to 672 and 733 to 757. Proteins S6, S8, S15 and S18 were shown to protect nucleotides 563 to 605, 624 to 680, 702 to 770, 818 to 839 and 844 to 891, which span the entire central domain of the 16 S rRNA molecule (nucleotides 560 to 890). The binding site for each protein contains helical elements as well as single-stranded internal loops ranging in size from a single bulged nucleotide to 20 bases. Three terminal loops and one stem-loop structure within the central domain of the 16 S rRNA were not protected in the four-protein complex. Interestingly, bases within or very close to these unprotected regions have been shown to be accessible to chemical and enzymatic probes in 30 S subunits but not in 70 S ribosomes. Furthermore, nucleotides adjacent to one of the unprotected loops have been cross-linked to a region near the 3' end of 16 S rRNA. Our observations and those of others suggest that the bases in this domain that are not sequestered by interactions with S6, S8, S15 or S18 play a role involved in subunit association or in tertiary interactions between portions of the rRNA chain that are distant from one-another in the primary structure.(ABSTRACT TRUNCATED AT 400 WORDS) 相似文献
4.
The acidic proteins B-L13 (homologous to Escherichia coli protein L7/L12) and B-L8, from the 50 S subunit of Bacillus stearothermophilus ribosomes, form a stable complex. Trypsin digestion of ribosomes generates an N-terminal fragment of B-L13 (approximately residues 1 to 47) which can associate with B-L8, displacing intact B-L13, and bind to B-L13-deficient ribosomes. Displacement of B-L13 from the B-L8 · B-L13 complex by the B-L13 N-terminal fragment causes a change in gel electrophoretic mobility of the complex, and titration of the complex with fragment indicates unambiguously that it contains four molecules of B-L13. Evidence is presented that B-L13 forms a dimer in solution, and that the dimer associates intact with B-L8. Reconstituted 50 S subunits in which B-L13 is replaced by its N-terminal fragment have the same functional properties as 50 S subunits missing B-L13 altogether: polypeptide synthesis is reduced but not abolished; ability to bind elongation factor EF-G and GTP is severely reduced; and peptidyl transferase activity and ability to associate with a 30 S subunit · Phe-tRNA · poly(U) complex are unaffected (relative to intact 50 S subunits). 相似文献
5.
D F Kimball L Peterson D J McLoughlin R G Wolfe 《Archives of biochemistry and biophysics》1979,195(1):66-73
Initial rate, product inhibition, and isotope rate kinetic studies of pig heart mitochondrial and supernatant malate dehydrogenases, acting upon the nonphysiological substrates, meso-tartrate and 2-keto-3-hydroxysuccinate, are reported. The measured spontaneous keto-enol equilibrium for 2-keto-3-hydroxysuccinate in 0.05 m Tris-acetate (pH 8.0) at 25 °C favors the enol form, dihydroxyfumarate, with an apparent equilibrium constant of 0.036. The enzyme-catalyzed reaction favors meso-tartrate with an apparent equilibrium constant of 1.25 × 10?6, M?1 at pH 8.0. The mechanism apparently remains ordered bi bi for both enzymes when these nonphysiological substrates are used, and the chemical-converting hydride transfer step becomes more rate limiting for both enzymes. This conclusion is supported by and values of 2.6 and 3.1, respectively, for the mitochondrial enzyme and 1.9 and 2.9, respectively, for the supernatant enzyme. 相似文献
6.
Electron spin resonance studies of the effects of lipids on the environment of proteins in mitochondrial membranes 总被引:1,自引:0,他引:1
G Lenaz G Curatola L Mazzanti G Zolese G Ferretti 《Archives of biochemistry and biophysics》1983,223(2):369-380
The physical state of mitochondrial membranes has been investigated by means of stearic acid spin labels and of a maleimide spin label covalently bound to protein sulfhydryl groups. Stearic acid spin labels 5-NS and 16-NS show that n-butanol enhances the lipid fluidity of mitochondrial membranes in the whole temperature range between 4 and 37 degrees C; the effects in the hydrophobic membrane core, probed by 16-NS, are already apparent at 10 mM butanol. In liposomes formed of mitochondrial phospholipids, a fluidizing effect appears only at much higher concentration. Such results are compatible with the idea that butanol destabilizes lipid-protein interactions. On the other hand, the ratio between weakly and strongly immobilized SH groups probed by maleimide spin label is only slightly affected in the temperature range of 4-37 degrees C by addition of high concentrations of n-butanol, indicating that the environments probed are stable to agents inducing fluidity changes in the lipids. There are, however, indications that the environment probed by maleimide is affected by lipids, since the spin label, when bound to lipid-depleted mitochondria, becomes more immobilized, reconstitution of such lipid-depleted membranes with phospholipids restores the original spectra. 相似文献
7.
The regulation of serine hydroxymethyltransferase, methylenetetrahydrofolate reductase, and methyltetrahydropteroylpolyglutamate:homocysteine methyltransferase was investigated in Neurospora crassa. Adding choline to the medium decreased the specific activity of these enzymes. Methionine potentiated the choline effect, but, when added alone, was without effect. Neither choline, methionine, nor S-adenosylmethionine appears to be the immediate corepressor of synthesis of these enzymes.Several nonallelic mutants were examined for the enzymes of methionine methyl group synthesis. The formate-requiring mutant for lacks serine hydroxymethyltransferase. The methionine-requiring mutants me-1 and me-8 lack, respectively, the reductase and the methyltransferase. The methionine-requiring mutants me-1, me-6 (folate polyglutamate synthetase deficient) and me-8 were found to have significantly higher serine hydroxymethyltransferase specific activities than did the wild-type strain. 相似文献
8.
The molecular weights of the proteins from rabbit liver ribosomal 40 S and 60 S subunits were determined after preliminary separation of these proteins by two-dimensional electrophoresis: each spot present in the polyacrylamide slab was cut off, eluted and rerun in a SDS one-dimensional polyacrylamide gel. The molecular weights range from 9,000 to 35,000 with a number-average molecular weight of 19,600 for the 40 S proteins, and from 9,400 to 52,000 with a number-average molecular weight of 23,600 for 60 S proteins. 相似文献
9.
Results of neutron-scattering experiments to determine the distances between seven pairs of proteins within the 30 S ribosomal subunit are presented. These results, combined with earlier data (Engelman et al., 1975; Moore et al., 1977) lead to the construction of a three-dimensional map of the positions of the centers of mass of proteins S3, S4, S5, S7, S8 and S9. The properties of this map and its relationship to other information on the structure of the 30 S subunit are discussed. 相似文献
10.
RNA splicing in Neurospora mitochondria. Defective splicing of mitochondrial mRNA precursors in the nuclear mutant cyt18-1 总被引:12,自引:0,他引:12
cyt18-1 (299-9) is a nuclear mutant of Neurospora crassa that has been shown to have a temperature-sensitive defect in splicing the mitochondrial large rRNA intron. In the present work, we investigate the effect of the cyt18-1 mutation on splicing of mitochondrial mRNA introns. Two genes were studied in detail; the cytochrome b (cob) gene, which contains two introns, and a "long form" of the cytochrome oxidase subunit I (coI) gene, which contains four introns. We found that splicing of both cob introns and splicing of at least two of the coI introns are strongly inhibited in the mutant, whereas splicing of coI intron 1, which is excised as a 2.6 X 10(3) base circle, is relatively unaffected. The rRNA intron and both cob introns are group I introns, whereas the circular coI intron may belong to another structural class. Control experiments showed that the degree of inhibition of splicing is greater in the mutant than can be accounted for by severe inhibition of mitochondrial protein synthesis. Finally, experiments in which mutant cells were shifted from 25 degrees C to 37 degrees C showed that splicing of the large rRNA precursor and splicing of the coI mRNA precursor are inhibited with similar kinetics. Considered together, our results suggest that the cyt18 gene encodes a trans-acting component that is required for the splicing of group I mitochondrial DNA introns or some subclass thereof. Since Neurospora cob intron 1 has been shown to be self-splicing in vitro, defective splicing of this intron in cyt18-1 indicates that an essentially RNA-catalyzed splicing reaction must be facilitated by a trans-acting factor, presumably a protein, in vivo. 相似文献
11.
Structural variations and optional introns in the mitochondrial DNAs of Neurospora strains isolated from nature 总被引:6,自引:0,他引:6
Mitochondrial DNAs from ten wild-type Neurospora crassa, Neurospora intermedia, and Neurospora sitophila strains collected from different geographical areas were screened for structural variations by restriction enzyme analysis. The different mtDNAs show much greater structural diversity, both within and among species, than had been apparent from previous studies of mtDNA from laboratory N. crassa strains. The mtDNAs range in size from 60 to 73 kb, and both the smallest and largest mtDNAs are found in N. crassa strains. In addition, four strains contain intramitochondrial plasmid DNAs that do not hybridize with the standard mtDNA. All of the mtDNA species have a basically similar organization. A 25-kb region that includes the rRNA genes and most tRNA genes shows very strong conservation of restriction sites in all strains. The 2.3-kb intron found in the large rRNA gene in standard N. crassa mtDNAs is present in all strains examined, including N. intermedia and N. sitophila strains. The size differences between the different mtDNAs are due to insertions or deletions that occur outside of the rRNA-tRNA region. Restriction enzyme and heteroduplex mapping suggest that four of these insertions are optional introns in the gene encoding cytochrome oxidase subunit I. Mitochondrial DNAs from different wild-type strains contain zero, one, three, or four of these introns. 相似文献
12.
13.
Identification of protein S 1 at the messenger RNA binding site of the Escherichia coli ribosome 总被引:5,自引:0,他引:5
I. Fiser K.H. Scheit G. Stöffler E. Kuechler 《Biochemical and biophysical research communications》1974,60(3):1112-1118
Poly-4-thiouridylic acid acts as messenger RNA for polyphenylalanine synthesis in an protein synthesizing system. When a complex consisting of ribosomes, poly-4-thiouridylic acid and Phe-tRNA is irradiated at 300 to 400 nm, covalent bonds between this messenger RNA and protein S 1 are formed. 相似文献
14.
Length polymorphisms, restriction site variation, and maternal inheritance of mitochondrial DNA of Drosophila melanogaster 总被引:6,自引:0,他引:6
We have studied the mitochondrial DNA in three wild type laboratory strains of Drosophila melanogaster, ry+5 and two Oregon R-substrains, called here R and E. Lengths of the restriction bands for EcoRI, BglII, HpaII, MspI, HaeIII, and HindIII were compared. The number of restriction sites was identical in all strains, with the exception of an extra HaeIII site in ry+5. Careful comparison of restriction fragment lengths showed that bands containing the AT-rich region were different in length among all strains. The laboratory strains, ry+5, proved to be a mixture of strains carrying different mtDNAs; these separated into substrains G1 and G2 in the progeny of single pair matings. Adult progeny of reciprocal crosses of G1 and R were analyzed by HaeIII restriction digestion. The results demonstrated maternal inheritance for both the extra restriction site and band containing the AT-rich region. 相似文献
15.
16.
Crosslinking of chemically reactive A-U-G analogs to ribosomal proteins within initiation complexes.
O. Pongs H.U. Petersen M. Grunberg-Manago E. Lanka R. Bald G. Stöffler 《Journal of molecular biology》1979,134(2):329-345
A-U-G analogs, either reactive on their 5′ or their 3′ side, were employed in affinity labeling of the ribosomal A-U-G binding site. These experiments have been carried out such that the chemically reactive A-U-G analog became covalently bonded to ribosomal proteins only in the presence of fMet-tRNAfMet and initiation factors. Subsequent radioimmunodiffusion of A-U-G-labeled proteins identified proteins IF3, S1, S18, S21 and L11 as being in the neighborhood of the ribosomal codon binding site. A location of reactive sites of these proteins relative to the P or A site bound codon is, however, not clear.The A-U-G labeling results are quantitatively as well as qualitatively very different in the absence or presence of fMet-tRNAfMet. It is concluded, therefore, that fMet-tRNAfMet directs A-U-G into its final binding site. Streptomycin cannot release fMet-tRNAfMet from initiation complexes which contain irreversibly bound 5′- {4-(bromoacetamido)phenylphospho}-adenylyl-(3′–5′)-uridylyl-(3′–5′)-guanosine. This suggests that codon-anticodon interaction between A-U-G and fMet-tRNAfMet is still intact in the P site of the ribosome. 相似文献
17.
C Nishio S Komura K Kurahashi 《Biochemical and biophysical research communications》1983,116(2):751-758
Biogenesis of subtilin, an antimicrobial peptide produced by Bacillus subtilis ATCC 6633, was studied in growing cells. Pulse-chase labeling experiments with [35S]cysteine revealed the presence of precursor proteins of subtilin. The synthesis of both precursor proteins and subtilin was inhibited by inhibitors of protein and RNA synthesis. When the precursor proteins were incubated with crude extracts of the organism in vitro, they were converted to subtilin. Pepstatin and phenylmethylsulfonyl fluoride in combination inhibited this conversion. 相似文献
18.
Vasopressin, angiotensin II, glucagon and epinephrine (through a cAMP-independent, alpha1adrenergic mechanism), stimulate ureogenesis in isolated rat hepatocytes. Mitochondria, isolated from hepatocytes which were previously treated with these hormones, displayed an enhanced rate of citrulline synthesis in the presence of NH4Cl as the nitrogen source. When mitochondria were incubated with glutamine as the nitrogen source, only those mitochondria isolated from hepatocytes previously treated with epinephrine or glucagon displayed an enhanced capacity to synthesize citrulline.When cells were incubated in the absence of extracellular calcium, the effects of vasopressin and angiotensin II on urea synthesis were abolished, whereas those of epinephrine and glucagon were only diminished. Mitochondria isolated from cells incubated under these conditions, showed that the effect of all these hormones on citrulline synthesis could still be observed. However, the effects of glucagon and epinephrine plus propranolol were larger than those of angiotensin II or vasopressin.Phosphatidylinositol labeling was significantly increased by epinephrine, vasopressin and angiotensin II both in the absence or presence of calcium. Cyclic AMP levels were significantly increased by glucagon or epinephrine but not by vasopressin or angiotensin II. The effect of epinephrine on cyclic AMP levels was blocked by propranolol both in the absence or presence of calcium. 相似文献
19.