Degradation of a specific 5 S RNA – 23 S RNA — protein complex by pancreatic ribonuclease

Gray, Peter N.; Bellemare, Guy; Monier, R.

doi:10.1016/0014-5793(72)80756-7

Cited by 28 publications

(7 citation statements)

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“…32P-labelled and unlabelled 5S RNA were prepared as described earlier from E. coli MRE600 or K12 RNase I10 (1). The RNA was renatured by heating at 55°C for 10 min in HMK reconstitution buffer (30 mM Hepes-OH, 20 mM MgCl2, 300 mM KCl, pH 7.6) and slowly cooling (15).…”

Section: Methodsmentioning

confidence: 99%

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A ribonuclease-resistant region of 5S RNA and its relation to the RNA binding sites of proteins L18 and L25

Douthwaite¹,

Garrett²,

Wagner³

et al. 1979

Nucl Acids Res

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An RNA fragment, constituting three subfragments of nucleotide sequences 1-11, 69-87 and 89-120, is the most ribonucleaseresistant part of the native 5S RNA of Escherichia coli, at 0°C. A smaller fragment of nucleotide sequence 69-87 and 90-110 is ribonuclease-resistant at 250.Degradation of the L25-5S RNA complex with ribonuclease A or T2 yielded RNA fragments similar to those of the free 5S RNA at 0°C and 25°C; moreover L25 remained strongly bound to both RNA fragments and also produced some opening of the RNA structure in at least two positions.Protein L18 initially protected most of the 5S RNA against ribonuclease digestion, at 0°C, but was then gradually released prior to the formation of the larger RNA fragment. It cannot be concluded, therefore, as it was earlier (Gray et al., 1973), that this RNA fragment contains the primary binding site of L18.

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Section: Methodsmentioning

confidence: 99%

“…Gray et al (1,2) first identified an RNA region which was "protected" by proteins L18 and L25 against ribonuclease A digestion. This region occurred within the sequences 1-11 and 69-120.…”

Section: Introductionmentioning

confidence: 99%

A ribonuclease-resistant region of 5S RNA and its relation to the RNA binding sites of proteins L18 and L25

Douthwaite¹,

Garrett²,

Wagner³

et al. 1979

Nucl Acids Res

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“…It is therefore likely (but remains to be proven) that the complexes at e = 1.35 g/ml contain ribonuclease-resistant RNA, poor in uridylic acid, associated with the proteins. Ribonuclease-resistant R N A from nuclear DNA-like RNA falls into two classes : polypurines, in particular poly(A) sequences (see [14] for review) and double-stranded fragments, some of them rich in (G -1 C) addition, when dealing with particles, some R N A sequences could be protected from ribonuclease by proteins as shown for ribosomal RNA [18,19], and might therefore also be found in the complex at e = 1.35 g/ml. Indeed, the experiment of Fig.7D shows that some proteins were relatively tightly bound to the ribonuclease-resistant RNA since the density of the 32P-labelled complexes was never above 1.50 g/ml.…”

Section: Discussionmentioning

confidence: 99%

Effects of Sodium Chloride and Pancreatic Ribonuclease on the Rat‐Brain Nuclear Particles: The Fate of the Protein Moiety

Stévenin

Jacob

1974

European Journal of Biochemistry

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Previous results on RNA-labelled nuclear particles were compatible with a folded ribonucleoprotein strand structure. In the present experiments, particles were labelled with tritiated amino acids or with tritiated amino acids and [32P]orthophosphate in vim; they were prepared by sonication of nuclei.1. Proteins were released progressively from the particles (e = 1.39 g/ml) as shown by the accumulation of material of e = 1.31 -1.35 g/ml after treatment with 0.25 M and 0.4 M NaCl. Concomitantly, high-density ribonucleoproteins were formed.2. Proteins released from the particles at various NaCl concentrations (up to 2 M) sedimented between 0 and 15 S. 30-S aggregates corresponding to "informofers" were never detected. Some proteins remained bound to the RNA even after treatment with 2 M NaCl.3. Large protein aggregates contaminated the smallest particles (up to 50-60 S) as shown by the sedimentation properties of the products of extensive ribonuclease digestion and the density of protein-labelled fractions. 4.About half of the protein released from the particles by pancreatic ribonuclease treatment had a low sedimentation coefficient (0-15 S) after formaldehyde fixation. The other half had higher polydisperse sedimentation coefficient, and possibly resulted from artificial cross-linking. A 30-S protein peak was not detected. 5.The material released from the particles by ribonuclease banded at e = 1.35 g/ml, a higher density than that of free proteins (p = 1.31 g/ml). Treatment with 1 M NaCl after ribonuclease digestion released free proteins, some of which were phosphoproteins (e = 1.31 g/ml) and ribonucleoproteins of higher density. It is suggested that these complexes contain ribonuclease-resistant RNA.These results confirm and complete our previous ones. They are not compatible with the informofer model of the monoparticle since 30-S protein aggregates could not be isolated after salt or ribonuclease treatment. They are consistent with the folded ribonucleoprotein strand model which predicts the sequential release of proteins.In a previous study [1,2] of the action of ribonuclease and salts on nuclear particles containing DNA like RNA we could not find any evidence in favor of the "informofer" model proposed by Samarina et al.[3] and Lukanidin et al. [4,5], according to which, the RNA was localized at the surface of a 30-S protein aggregate, the informofer, stable at high salt concentration. On the contrary, our results were compatible with a folded ribonucleoprotein strand model. However, no direct proof for such a structural organization where only a fraction of the RNA sequences would be external could be furnished.Since our previous experiments [1,2] had only been performed with particles containing labelled RNA, so that the fate of the protein moeity was not followed, the evidence against the informofer was indirect. This paper reports results obtained with particles labelled either on their protein, or on both their RNA and protein moieties. METHODS Labelling of Par tidesIn order to obtain highly labelle...

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“…Although it is not yet known whether this region mediates in the binding of the renatured form, it is tempting to suppose that in this region the secondary structures of the denatured and native forms differ but are similar in the native and renatured forms. It is therefore of interest that it can be folded to give two hairpin-looped helical regions with a total of 11 base pairs [20]. The heating of native or renatured 5-5 RNA in the presence of low or moderate concentrations of NaCl in the absence of Mg2+ ions leads to a partial denaturation (unpublished results).…”

Section: (3)mentioning

confidence: 99%

The Kinetics of Renaturation of 5‐S RNA from Escherichia coli in the Presence of Mg²⁺ Ions

Richards

Lecanidou

Geroch

1973

European Journal of Biochemistry

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The kinetics of renaturation of denatured 5-S RNA from Escherichia coli have been studied by estimating the composition of reaction mixtures from absorbance profiles of stained zones containing the two forms separated by polyacrylamide-gel electrophoresis. The kinetics are first order and proceed to completion irrespective of temperature or Mga+ ion concentration, but they depend strongly on temperature. Linear Arrhenius plots yield an activation energy of 65 kcal/mol RNA. It is suggested that about nine base pairs of the denatured form dissociate and about the same number reform in a different configuration in the renatured conformer. Aubert et al. [5] have shown that 5-S RNA can exist in a t least two forms. These may be separated by chromatography on Sephadex G-100 or methylated bovine-serumalbumin-coated Kieselguhr [5] or electrophoretically on polyacrylamide gel [6] (and unpublished results). The form released from the 50-5 ribosomal subunit can participate in reconstitution, is termed the native form and can be partially converted to a denatured form by treatment with urea and EDTA [5]. The denatured form is chromatographically and electrophoretically distinct from the native form and has different spectroscopic properties [5--81. It is not a single conformational species since it may be resolved by polyacrylamide-gel electrophoresis into two components (unpublished results). On treatment with Mgz+ ions a t 60°C the denatured form is converted to a renatured form which is chromatographically and electrophoretically indistinguishable from the native form; it has closely similar optical properties [S] and can participate in the reconstitution of 50-8 ribosomal subunits [5]. Nevertheless it is claimed that the native and renatured forms are not identical in that the activity of the renatured form in reconstitution experiments is less than that of the native form [5] and that small differences in their optical properties ace discernible [S]. Jordan [9]has investigated the secondary structure of the native and denatured forms by partial digestion with nucleases and shown that the patterns of oligonucleotides produced are very different. It was concluded that the points of initial attack by the enzymes were not the same and inferred that the two forms had different secondary structures or base-pairing schemes. These observations do not preclude the possibility that they also differ in their tertiary structure. The renatured form has not yet been studied in this manner but in so far as the native and renatured forms are closely similar in many of their properties it is very likely that the denatured and renatured forms also differ in their secondary, and perhaps tertiary, structure.We have exploited, for the purposes of this investigation, the ability of polyacrylamide-gel electrophoresis to resolve the denatured and renatured [6] (and unpublished results) forms in order to study the effect of temperature and Mg2+ ion concentration on the rate of renaturation. This technique is particularly useful in this con...

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Degradation of a specific 5 S RNA – 23 S RNA — protein complex by pancreatic ribonuclease

Cited by 28 publications

References 4 publications

A ribonuclease-resistant region of 5S RNA and its relation to the RNA binding sites of proteins L18 and L25

A ribonuclease-resistant region of 5S RNA and its relation to the RNA binding sites of proteins L18 and L25

Effects of Sodium Chloride and Pancreatic Ribonuclease on the Rat‐Brain Nuclear Particles: The Fate of the Protein Moiety

The Kinetics of Renaturation of 5‐S RNA from Escherichia coli in the Presence of Mg²⁺ Ions

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Degradation of a specific 5 S RNA – 23 S RNA — protein complex by pancreatic ribonuclease

Cited by 28 publications

References 4 publications

A ribonuclease-resistant region of 5S RNA and its relation to the RNA binding sites of proteins L18 and L25

A ribonuclease-resistant region of 5S RNA and its relation to the RNA binding sites of proteins L18 and L25

Effects of Sodium Chloride and Pancreatic Ribonuclease on the Rat‐Brain Nuclear Particles: The Fate of the Protein Moiety

The Kinetics of Renaturation of 5‐S RNA from Escherichia coli in the Presence of Mg2+ Ions

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The Kinetics of Renaturation of 5‐S RNA from Escherichia coli in the Presence of Mg²⁺ Ions