The structure of RNA. Reovirus RNA and transfer RNA have similar three-dimensional structures, which differ from DNA.
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Reproducible conditions have been developed for crystallization of transfer RNA. The conditions may be applicable to many pure transfer RNA species since identical procedures (except for initial transfer-RNA concentration) yielded good crystals from both yeast and Escherichia coli transfer RNA. These crystals, which must be kept at temperatures below about 10 degrees C and handled in vapor of controlled alcohol concentration, have been studied by x-ray crystallography. The availability of crystals of a nucleic acid opens a route for extending knowledge of the tertiary structure of transfer RNA and its relation to important biological functions.
A continuous spectrophotometric assay is described for the aminoacylation reaction catalyzed by Escherichia coli alanyl-transfer RNA synthetase. The assay is based on coupling the alanyl-tRNA synthetase-dependent formation of AMP to the lactate dehydrogenase oxidation of NADH. Oxidation of NADH, as monitored at 340 nm, is shown to be stoichiometric with the formation of alanyl-tRNA(Ala). This assay will facilitate the rapid accumulation and analysis of kinetic data for alanyl-tRNA synthetase and should be applicable to aminoacyl-tRNA synthetases in general.
Escherichia coli ribosomes with a G to C transversion at position 2661 in 23 S ribosomal RNA are more accurate in tRNA selection than wild-type ribosomes. This enhanced accuracy is due to improved initial selection of ternary complexes rather than proofreading of aminoacyl tRNAs. The 2661C mutation reduces the binding rate of cognate ternary complexes to the A-site. This binding rate deficiency becomes dramatic when ribosomes also harbour an S12 mutation with a streptomycin-resistant, hyperaccurate phenotype. In this case, severe loss of kinetic efficiency in EF-Tu function leads to cell death. Streptomycin restores viability by increasing the association rate of ternary complex to these doubly altered ribosomes. The binding rate of EF-G to 2661C ribosomes is also reduced while the translocation rate is unaffected.
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Animalization (ectodermalization) of embryos of Arbacia punctulata was induced by continuous treatment from the 2-cell stage with Evans Blue. Quantitative measurements of rates of accumulation of 5S RNA and tRNA in mesenchyme blastulae, in plutei and in corresponding animalized embryos showed that these rates per embryo and per cell were similar in control and in animalized embryos. The alterations in normal cell interactions and germ layer formation induced by Evans Blue did not affect the synthesis of 5S RNA and tRNA in sea urchin embryos.
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The rates of synthesis of ribosomes, 5S RNA, and tRNA necessary to maintain the steady-state concentrations of these entities in liver cytoplasm of adult rats were determined. On the average, each liver cell in the adult rat synthesizes 650 ribosomes, 650 molecules of 5S RNA, and 11,000 molecules of tRNA each minute. The numbers of genes per liver cell for rRNA, 5S RNA, and tRNA were 330, 1660, and 13,000, respectively, as determined by RNA: DNA hybridization experiments. Thus, on the average, individual genes for rRNA, tRNA, and 5S RNA are transcribed twice a minute, once a minute, and once every 2.5 minutes, respectively, in the adult rat liver.
Total Escherichia coli RNA has been fractionated on Sepharose 6B in 0.1 M ammonium acetate, pH 5.0. The elution order was 23S rRNA, 16S rRNA,, 5S rRNA, and tRNA, which is in contrast with the reported elution order of eukaryotic RNA, chromatographed under similar conditions. tRNA was obtained in two regions, well separated from the high molecular weight rRNA but with a slight contamination of 5S rRNA. The capacity is at least 40 A260 units of RNA per ml of gel.
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In the present communication a characterization of the 5 S rRNA genes and the tRNA genes of Tetrahymena pyriformis has been performed. The number of 5 S rRNA and tRNA genes in the macromolecular DNA has been established. Furthermore no sequence homology is observed for these genes. The number of both types of genes does not change significantly under starvation conditions. The genomic organization of the 5 S rRNA and tRNA genes has been investigated. From in vivo replication studies it is concluded, that replication of both 5 S rRNA and tRNA genes takes place throughout the whole S-period.
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