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The origin of the polydispersity in sedimentation patterns of rapidly labelled nuclear ribonucleic acid.

ABSTRACT: 1. A study was made of the sedimentation properties of purified preparations of the rapidly labelled RNA in the nucleus and the cytoplasm of the HeLa cell. The sedimentation of the rapidly labelled nuclear RNA was very sensitive to changes in ionic strength and bivalent cation concentration. Under the conditions usually used in sucrose-density-gradient centrifugation the rapidly labelled nuclear RNA showed extreme polydispersity, and much of it sedimented more rapidly than the 28s RNA. At low ionic strength and after removal of Mg(2+), however, the rapidly labelled nuclear RNA sedimented as a single peak at about 16s. The conversion of the polydisperse material into the 16s form did not involve degradation of the RNA, since the effect could be reversed by increasing the ionic strength of the solution. 2. The cytoplasm did not contain any RNA that showed polydisperse sedimentation under the usual conditions of sucrose-density-gradient centrifugation, or that had the same sensitivity as the rapidly labelled nuclear RNA to changes in ionic strength. All the radioactivity in the cytoplasmic RNA sedimented with the 28s, 16s and 4s components over a wide range of physical conditions, but these components did contain a labelled fraction with some of the features of the rapidly labelled nuclear RNA on columns of methylated albumin on kieselguhr. 3. In both nucleus and cytoplasm the RNA detected by ultraviolet absorption could also be converted into a 16s form by removal of bivalent cations at low ionic strength; this effect was again, within certain limits, reversible. The nuclear RNA as a whole was more susceptible to changes in ionic strength than the cytoplasmic RNA. 4. It thus appears that all the RNA in the cell, except the 4s RNA, can be prepared, without degradation, as a single peak sedimenting at about 16s. The relationship of these various 16s components to each other is discussed.

SUBMITTER: Bramwell ME

PROVIDER: S-EPMC1270489 | biostudies-other | 1967 Jun

REPOSITORIES: biostudies-other

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Project description:1. Rapidly labelled RNA from Escherichia coli K 12 was characterized by hybridization to denatured E. coli DNA on cellulose nitrate membrane filters. The experiments were designed to show that, if sufficient denatured DNA is offered in a single challenge, practically all the rapidly labelled RNA will hybridize. With the technique employed, 75-80% hybridization efficiency could be obtained as a maximum. Even if an excess of DNA sites were offered, this value could not be improved upon in any single challenge of rapidly labelled RNA with denatured E. coli DNA. 2. It was confirmed that the hybridization technique can separate the rapidly labelled RNA into two fractions. One of these (30% of the total) was efficiently hybridized with the low DNA/RNA ratio (10:1, w/w) used in tests. The other fraction (70% of the total) was hybridized to DNA at low efficiencies with the DNA/RNA ratio 10:1, and was hybridized progressively more effectively as the amount of denatured DNA was increased. A practical maximum of 80% hybridization of all the rapidly labelled RNA was first achieved at a DNA/RNA ratio 210:1 (+/-10:1). This fraction was fully representative of the rapidly labelled RNA with regard to kind and relative amount of materials hybridized. 3. In competition experiments, where additions were made of unlabelled RNA prepared from E. coli DNA, DNA-dependent RNA polymerase (EC 2.7.7.6) and nucleoside 5'-triphosphates, the rapidly labelled RNA fraction hybridized at a low (10:1) DNA/RNA ratio was shown to be competitive with a product from genes other than those responsible for ribosomal RNA synthesis and thus was presumably messenger RNA. At higher DNA/rapidly labelled RNA ratios (200:1), competition with added unlabelled E. coli ribosomal RNA (without messenger RNA contaminants) lowered the hybridization of the rapidly labelled RNA from its 80% maximum to 23%. This proportion of rapidly labelled RNA was not competitive with E. coli ribosomal RNA even when the latter was in large excess. The ribosomal RNA would also not compete with the 23% rapidly labelled RNA bound to DNA at low DNA/RNA ratios. It was thus demonstrated that the major part of E. coli rapidly labelled RNA (70%) is ribosomal RNA, presumably a precursor to the RNA in mature ribosomes. 4. These studies have shown that, when earlier workers used low DNA/RNA ratios (about 10:1) in the assay of messenger RNA in bacterial rapidly labelled RNA, a reasonable estimate of this fraction was achieved. Criticisms that individual messenger RNA species may be synthesized from single DNA sites in E. coli at rates that lead to low efficiencies of messenger RNA binding at low DNA/RNA ratios are refuted. In accordance with earlier results, estimations of the messenger RNA content of E. coli in both rapidly labelled and randomly labelled RNA show that this fraction is 1.8-1.9% of the total RNA. This shows that, if any messenger RNA of relatively long life exists in E. coli, it does not contribute a measurable weight to that of rapidly labelled messenger RNA.

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The origin of the polydispersity in sedimentation patterns of rapidly labelled nuclear ribonucleic acid.

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