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The distribution of ribosomal ribonucleic acids among subcellular fractions from bacteria and the adverse effect of the membrane fraction on the stability of ribosomes.

ABSTRACT: 1. The distributions of nucleic acids and protein among fractions obtained by differential centrifugation from species of Pseudomonas, Aerobacter, Escherichia, Proteus and Bacillus have been studied. 2. The DNA in a cell wall-membrane fraction obtained by low-speed centrifugation from the Gram-negative species could be removed by homogenizing and subsequent washing. About 7-14% of the total RNA remained firmly attached and resembled ribosomal RNA in base composition. A similar fraction from the Gram-positive B. subtilis contained about one-half of the total bacterial DNA and only 60% of this could be removed by homogenizing and subsequent washing. 3. A deposit obtained by high-speed centrifugation could be separated into a heavy ribosome layer and a light turbid layer. In E. coli B the latter contained about equal concentrations of RNA and DNA and accounted for about one-half of the total bacterial NADP-activated 6-phosphogluconate dehydrogenase. 4. The washed cell wall-membrane fraction from most species accelerated the degradation of ribosomes. In Pr. vulgaris the activity of this fraction was exceptionally high and resulted in the progressive degradation of ribosomes during their isolation from this species. 5. A possible connexion between ribosome degradation and the synthesis of flagella is discussed in the light of these results.

SUBMITTER: Wade HE

PROVIDER: S-EPMC1207213 | biostudies-other | 1965 Sep

REPOSITORIES: biostudies-other

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Differential stability of 28s and 18s rat liver ribosomal ribonucleic acids.

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Intranuclear maturation pathways of rat liver ribosomal ribonucleic acids.

Project description:The maturation of pre-rRNA (precursor to rRNA)in liver nuclei is studied by agar/ureagel electrophoresis, kinetics of labelling in vivo with [14C] orotate and electron-microscopic observation of secondary structure of RNA molecules. (1) Processing starts from primary pre-rRNA molecules with average mol. wt. 4.6X10(6)(45S) containing the segments of both 28S and 18S rRNA. These molecules form a heterogeneous peak on electrophoresis. The 28S rRNA segment is homogeneous in its secondary structure. However, the large transcribed spacer segment (presumably at the 5'-end) is heterogeneous in size and secondary structure. A minor early labelled RNA component with mol.wt. about 5.8X10(6) is reproducibly found, but its role as a pre-rRNA species remains to be determined. (2) The following intermediate pre-rRNA species are identified: 3.25X10(6) mol.wt.(41S), a precursor common to both mature rRNA species ; 2.60X10(6)(36S) and 2.15X10(6)(32S) precursors to 28S rRNA; 1.05X10(6) (21S) precursor to 18S rRNA. The pre-rRNA molecules in rat liver are identical in size and secondary structure with those observed in other mammalian cells. These results suggest that the endonuclease-cleavage sites along the pre-rRNA chain are identical in all mammalian cells. (3) Labelling kinetics and the simultaneous existence of both 36S and 21S pre-rRNA reveal that processing of primary pre-rRNA in adult rat liver occurs simultaneously by at least two major pathways: (i) 45S leads to 41S leads to 32S+21S leads to 28S+18S rRNA and (ii) 45S leads to 41S leads to 36S+18S leads to 32S leads to 28S rRNA. The two pathways differ by the temporal sequence of endonuclease attack along the 41 S pre-rRNA chain. A minor fraction (mol.wt.2.9X10(6), 39S) is identified as most likely originating by a direct split of 28S rRNA from 45S pre-rRNA. These results show that in liver considerable flexibility exists in the order of cleavage of pre-rRNA molecules during processing.

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Project description:The biosynthesis and stability of various RNA fractions was studied in RC(str) and RC(rel) multiple amino acid auxotrophs of Escherichia coli. In conditions of amino acid deprivation, RC(str) mutants were labelled with exogenous nucleotide bases at less than 1% of the rate found in cultures growing normally in supplemented media. Studies by DNA-RNA hybridization and by other methods showed that, during a period of amino acid withdrawal, not more than 60-70% of the labelled RNA formed in RC(str) mutants had the characteristics of mRNA. Evidence was obtained for some degradation of newly formed 16S and 23S rRNA species to heterogeneous material of lower molecular weight. This led to overestimations of the mRNA content of rapidly labelled RNA from such methods as simple examination of sucrose-density-gradient profiles. In RC(rel) strains the absolute and relative rates of synthesis of the various RNA fractions were not greatly affected. However, the stability of about half of the mRNA fraction was increased in RC(rel) strains during amino acid starvation, giving kinetics of mRNA labelling and turnover that were identical with those found in either RC(str) or RC(rel) strains inhibited by high concentrations of chloramphenicol. Coincidence hybridization techniques showed that the mRNA content of amino acid-starved RC(str) auxotrophs was unchanged from that found in normally growing cells. In contrast, RC(rel) strains deprived of amino acids increased their mRNA content about threefold. In such cultures the mRNA content of accumulating newly formed RNA was a constant 16% by wt.

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