A study of the alkaline hydrolysis of fractionated reticulocyte ribosomal ribonucleic acid and its relevance to secondary structure

Cox, Robert A.; Gould, Hannah; Kanagalingam, K.

doi:10.1042/bj1060733

Cited by 31 publications

(13 citation statements)

References 17 publications

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“…When RNA was heated to 950 in 1% formaldehyde the formation ofeven very short double-helical segments was prevented. It was found that the reversible hypochromism of RNA in 1% formaldehyde was scarcely dependent on chain length over the range 2*7-30s whereas the hypochromism found in the absence of formaldehyde was profoundly affected by degradation from 30s to 2-7s RNA (Cox et al 1968). Hypochromism due to 'stacking' between unpaired residues is known to be only slightly dependent on chain length (e.g.…”

Section: Discussionmentioning

confidence: 99%

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A spectrophotometric study of the denaturation of deoxyribonucleic acid in the presence of urea or formaldehyde and its relevance to the secondary structure of single-stranded polynucleotides

Cox

Kanagalingam

1968

Biochemical Journal

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1. The thermal denaturation of DNA from rat liver was studied spectrophotometrically. In sodium phosphate buffers denaturation led to a single-stranded form having, at 25 degrees , about 25% of the hypochromism of the intact double helix. 2. The hypochromism of the denatured form was the same in 1mm- as in 10mm-sodium phosphate buffer and was scarcely affected by reaction with formaldehyde. The hypochromism was decreased by about 40% in the presence of 8m-urea. 3. The hypochromism of denatured DNA at low ionic strengths was about the same as that of fragments of reticulocyte ribosomal RNA that were too short to form double-helical secondary structure and about the same as that of RNA after reaction with formaldehyde. 4. The spectrum of DNA was slightly affected by the presence of 8m-urea or 4m-guanidinium chloride. The differences in the spectrum of the native and denatured forms of DNA in 0.1m-sodium phosphate buffer, in 8m-urea-10mm-sodium phosphate buffer and in 4m-guanidinium chloride-10mm-sodium phosphate buffer, pH7.6, were similar but not identical. 5. Denatured rat liver DNA appears to have no double-helical character at 25 degrees in 10mm-sodium phosphate buffer, pH7.6; increasing the buffer concentration to 0.1m leads to a more compact form in which about 40% of the residues form base pairs.

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Section: Discussionmentioning

confidence: 99%

“…RNA was degraded either with alkali (Cox, Gould & Kanagalingam, 1968) or with ribonuclease T1 (Gould, 1967). The fragments were fractionated according to size either by chromatography on Sephadex G-100 or by polyacrylamide-gel electrophoresis.…”

Section: P0cmentioning

confidence: 99%

A spectrophotometric study of the denaturation of deoxyribonucleic acid in the presence of urea or formaldehyde and its relevance to the secondary structure of single-stranded polynucleotides

Cox

Kanagalingam

1968

Biochemical Journal

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“…Although it is still not established that the secondary structures present in the degraded fragments are identical to those in the intact rRNA, it is very likely that this is so. The average size of hairpin loops with helical structure has been estimated to be about 25 nucleotides for the rRNA of the smaller ribosomal subunit and 35 for the rRNA of the larger ribosomal subunit of rabbit reticulocytes (49).…”

Section: Secondary Structure Of Rrna In Isolated Statesmentioning

confidence: 99%

Bacterial Ribosome

Nomura¹

1970

Bacteriological Reviews

102

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“…The molecular weight of fraction I1 was 15700 & 1800, corresponding to 49 f 6 nucleotide bases. This figure may be compared with an estimate of 35 f 7 for the number of bases per hairpin loop in the large subunit of reticulocyte ribosomes [17]. Thus, the structured region of the RNA of ribosomes from several different sources appear to be similar in size.…”

Section: Discussionmentioning

confidence: 96%

Partial Enzymic Digestion of 50 S Ribosomal Subunits from Escherichia coli

Spencer

Walker

1971

European Journal of Biochemistry

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50 S ribosomal subunits from Escherichia COG were incubated with an endonuclease specific for single-stranded nucleic acid. After incubation the sedimentation coefficient remained unaltered but gel filtration showed that loo/, of the nucleic acid in the ribosome had been degraded to small fragments. When the enzyme-treated native subunits were subsequently unfolded in EDTA their sedimentation properties were similar to controls. Thermal denaturation in the presence of formaldehyde produced small fragments of RNA with a sedimentation coefficient of 2s.Subunits which had fist been unfolded in EDTA and then incubated with enzyme were rapidly degraded to small fragments with a sedimentation coefficient of 2 S. The degraded fragments had 20 Ol0 hyperchromism on thermal denaturation which showed that the secondary structure of the RNA had remained intact. These experiments were interpreted in terms of the hairpin-loop model of RNA. They show that on the surface of the native subunit there is no interhelical single-stranded RNA which is accessible to enzyme whereas there is a significant amount of intrahelical single-stranded RNA. In the unfolded subunit the interhelical regions become exposed to attack by the enzyme.A digest of unfolded subunits was separated by gel atration on Sephadex 6-100 into three fractions. Fraction I, which eluted at the exclusion volume of the column, was mainly aggregated protein. Fraction 11, which contained 65 of the ultraviolet absorbing material and no protein, was hyperchromic and the circular dichroic spectrum was consistent with this fraction having double-helical secondary structure. Fraction I1 moved as a single, sharp boundary on sedimentation with sz0 = 1.7 S and had a molecular weight of 15700. Fraction 111, which contained 3501, of the ultraviolet absorbing material and no protein, was low molecular weight nucleotide with no secondary structure.Ribosomal RNA is thought to consist of short regions of double-helical "hairpin loops" connected by single-stranded interhelical regions [l -31. I n the ribosome, the size of the hairpins, the size of the singlestranded regions, the position of attachment of the proteins, and the way in which the ribonucleoprotein strand is folded in the native ribosome is largely unknown, although various models have been proposed [4-61. Useful information about the tertiary structure of spherical viruses and ribosomes has been obtained by studying the susceptibility of the native nucleoprotein to enzymic digestion [7-lo]. These studies have usually used pancreatic RNAse. However, degradation of ribosomal RNA with this enzyme leads eventually to a loss of the secondary structure which introduces ambiguities in the interpretation of the results, especially in the case of ribosomes [18]. A n attempt has been made to gain more precise information about the arrangement of RNA in the 50s subunit by studying the action of an endonuclease, highly specific for single-stranded RNA, on native subunits and subunits which have been unfolded in EDTA [Ill. Analytical...

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A study of the alkaline hydrolysis of fractionated reticulocyte ribosomal ribonucleic acid and its relevance to secondary structure

Cited by 31 publications

References 17 publications

A spectrophotometric study of the denaturation of deoxyribonucleic acid in the presence of urea or formaldehyde and its relevance to the secondary structure of single-stranded polynucleotides

A spectrophotometric study of the denaturation of deoxyribonucleic acid in the presence of urea or formaldehyde and its relevance to the secondary structure of single-stranded polynucleotides

Bacterial Ribosome

Partial Enzymic Digestion of 50 S Ribosomal Subunits from Escherichia coli

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