On Charge and Specific Ion Effects on Sedimentation in the Ultracentrifuge

Pedersen, Kai O.

doi:10.1021/j150568a028

Cited by 113 publications

(28 citation statements)

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“…4 shows the standard sedimentation coefficient distributions of HT-DNA in 100.0 mM LiCl, NaCl, KCl and CsCl solutions. As reported by 79 Thus, with the combination of these effects mentioned above, we have s CsCl 4 s KCl 4 s NaCl 4 s LiCl with the same salt concentration of 100.0 mM. A significant difference in s 20,w in the presence of different salts is observed, which will be discussed as follows.…”

Section: Resultsmentioning

confidence: 54%

The effects of monovalent metal ions on the conformation of human telomere DNA using analytical ultracentrifugation

Gao

2016

Soft Matter

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A human telomere DNA segment (HT-DNA) can fold into a G-quadruplex in the presence of some monovalent cations. These cations can interact with the phosphate groups of the DNA segment and/or with the O6 oxygen atom of guanines, which are called non-specific interactions and specific interactions, respectively. However, until now how these two interactions affect the structure of HT-DNA has not been well understood. In this study, a combination of analytical ultracentrifugation (AUC) and circular dichroism (CD) was used to explore the effects of these two interactions on the structure of a 22-mer single-stranded DNA with a sequence of 5'-AGGG(TTAGGG)3-3'. The results showed that the standard sedimentation coefficient (s20,w) of HT-DNA starts to increase when the concentration of potassium ions (CK(+)) is higher than 10.0 µM due to the formation of a G-quadruplex through specific interactions. Whereas, for a control DNA, a higher CK(+) value of 1.0 mM was needed for increasing s20,w due to non-specific interactions. Moreover, potassium ions could promote the formation of the G-quadruplex much more easily than lithium, sodium and cesium ions, presumably due to its appropriate size in the dehydrated state and easier dehydration. The molar mass of DNA at different cation concentrations was nearly a constant and close to the theoretical value of the molar mass of monomeric HT-DNA, indicating that what we observed is the structural change of individual DNA chains.

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Section: Resultsmentioning

confidence: 54%

The effects of monovalent metal ions on the conformation of human telomere DNA using analytical ultracentrifugation

Gao

2016

Soft Matter

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“…The higher apparent specific volume for the 23 S rRNA in Recon Buffer may be explained by the presence of high concentration of ammonium ions. Since the partial specific volume for ammonium ion (tl .O ml g-') is more than four times the value for potassium ion (to.225 ml g-') [20,21], replacing the potassium ion with the ammonium ion may possibly result in an increase in the apparent partial specific volume of the salt-associated RNA. In any case it is apparent that there is a considerable difference in the apparent specific volumes which, if the gross conformations of the RNA in the two buffer systems are really identical, implies that there is a substantial change in the solvent-biopolymer interaction.…”

Section: Resultsmentioning

confidence: 99%

Physical characteristics of 23 S rRNA from the 50 S ribosomal subunit of escherichia coli

1981

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The in vitro reconstitution of the bacterial 30 S ribosomal subunits [l-2] and subsequently the 50 S subunits [3-51, has establishedthe fact that the information required for the assembly of the subunits is present in the structure of the proteins and/or RNAs. Previous work has also shown that the reconstitution process requires a rigid set of buffer conditions.Recently, Vasiliev and Zalite [6], using electron microscopy, concluded that the 23 S rRNA molecules are capable of acquiring a specific compact conformation which is morphologically close to intact 50 S subunits. However, their RNA was prepared in the presence of EtOH and spermidine, which have been shown to cause conformational changes on bacterial ribosomes [7,8]. Sieber et al. [9] also reported an electron microscopic analysis of the 23 S rRNA, which has an overall diameter larger than the dimensions reported by Vasiliev and Zalite 161, as well as the 50 S subunit [lo]. From these disparate results it is apparent that further physical studies are needed to clarify the structure of 23 S rRNA.We report here the physical characteristics of the 23 S rRNA under buffer conditions used for the formation of the first reconstitution intermediate, aswell as under low salt conditions normally used with hydrodynamic methods. We have compared these results with the hydrodynamic properties obtained previously on the first reconstitution intermediateOur results indicate that under reconstitution conditions, the conformation of the 23 S rRNA is considerably more extended than that found in the 50 S subunit, and that upon addition of protein the RNA structure does not appear to be appreciably affected. ElsevierlNorth-Holland Biomedical Press Materials and methods .l . Preparation of 50 S subunitsRibosomal subunits were prepared using previously described methods [12]. The 50 S and 30 S subunits were separated on a lo-30% exponential sucrose gradient formed in a Ti-15 Beckman zonal rotor spun at 32 000 rev./min for 14.5 h. The 50 S subunits were recovered from the sucrose fractions by precipitating with 2 volumes of ethanol after raising the Mg2+ and dithiothreitol concentration to 0.01 M and 0.001 M. The precipitate was pelleted by centrifugation at 20 000 X g for 30 min, dissolved and dialyzed at 4°C against 0.01 M Tris-HCl, pH 7.6,O.Ol M Mg(OAC)2, 0.06 M NH&l and 0.004 M fl-mercaptoethanol overnight. The purity of the ribosomal preparations was checked routinely by means of sedimentation velocity in the Beckman Model E analytical ultracentrifuge. 2. Preparation of 23 S rRNAThe 23 S and 5 S rRNAs were extracted from the 50 S subunits with phenol according to the method of Nierhaus et al. [5]. After the final extraction, the rRNA samples were precipitated with 2 volumes of EtOH, 20 mM Tris-HCl, pH 7.8, and pelleted at 10 000 revjmin for 30 min at 4°C in an HB-4 rotor. The pellets were dissolved in and dialyzed against 0.01 M Tris-HCI, pH 7.4,O.l M KC1 and 0.0015 M MgClz (Buffer I). The 23 S rRNA was separated from the 5 S rRNA by passing through a lo-30% exponentia...

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“…Experiments performed with PO K yelectrolytes without weights. Secondary salt effects (Pedersen, 1958) an c f specific binding of polydisperse extracts, a better way of obtaining them would 1 e by the the sedimentation ve P oci9 technique. extracts Prelimina?l quic ly established that, because of their high degree of polygelpermeation c E romatography, a single extract was subdivided into dispersity is much greater than might be deduced by comparing t K e widths of the final peaks with those obtained from earlier stages of the fractionation.…”

Section: Resultsmentioning

confidence: 99%

Molecular Weight and Shape of Humic Acid From Sedimentation and Diffusion Measurements on Fractionated Extracts

Cameron¹,

Thornton²,

Swift³

et al. 1972

Journal of Soil Science

155

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Summary Whole humic acid extracts are usually too polydisperse for reliable molecular‐weight measurement to be made in the ultracentrifuge by the sedimentation velocity technique. Consequently, the humic acid used in this study was fractionated with respect to molecular weight into fractions of low polydispersity by extensive use of gel‐permeation chromatography and other fractionation techniques. The sedimentation and diffusion coefficients of the fractions were determined and molecular‐weight values calculated. These values ranged approximately from 2 × 103 to 1.5 × 106, the higher figure not necessarily representing the upper limit for these substances. On the basis of the frictional parameters calculated from the experimental data, it is proposed that the molecule adopts the solution conformation of a randomly coiled polymer in which branching may be significant, particularly at higher molecular weights.

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On Charge and Specific Ion Effects on Sedimentation in the Ultracentrifuge

Cited by 113 publications

References 0 publications

The effects of monovalent metal ions on the conformation of human telomere DNA using analytical ultracentrifugation

The effects of monovalent metal ions on the conformation of human telomere DNA using analytical ultracentrifugation

Physical characteristics of 23 S rRNA from the 50 S ribosomal subunit of escherichia coli

Molecular Weight and Shape of Humic Acid From Sedimentation and Diffusion Measurements on Fractionated Extracts

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