Binding of 50 S ribosomal subunit proteins to 23 S RNA of Escherichia coli

Garret, R A.; Müller, Shirley A.; Spierer, Pierre; Zimmermann, Ra

doi:10.1016/0022-2836(74)90503-8

Cited by 61 publications

(19 citation statements)

References 13 publications

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“…It follows that the likelihood of artefacts being introduced by a protein 'wandering' from one RNA-protein complex to another during the course of isolation is very low. Proteins L2, L6 and L16 are 'binding' proteins [33], and all three have been shown to be capable of binding to the 18-S fragment which arises from the 3' end of the 23-S RNA [20]. On the other hand, L13 was also shown by the same series of experiments to bind exclusively to the 18-S RNA, whereas we find it distributed equally in both RNA fractions, suggesting that in the intact 50-S subunit L13 has contacts in both halves of the 23-S RNA.…”

Section: Analysis Of 50-s Subunit Rna Regionsmentioning

confidence: 99%

The Use of Formaldehyde in RNA‐Protein Cross‐linking Studies with Ribosomal Subunits from Escherichia coli

Möller

Rinke

Ross

et al. 1977

European Journal of Biochemistry

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Radioactive subunits from Escherichia coli ribosomes were treated with formaldehyde, with a view to inducing a controlled cross-linking reaction between protein and ribosomal RNA. Under the conditions described, about 10-15 of the total protein remained bound to the RNA in the presence of dodecyl sulphate, and little or no irreversible protein-protein cross-linking was observed. The RNA-protein complexes are of a rather unstable nature, and the reaction is spontaneously reversible.The proteins involved in the cross-linked complexes were examined by polyacrylamide gel electrophoresis, after removal of unbound protein and nuclease treatment to digest the RNA-protein complex. This showed that the individual ribosomal proteins were cross-linked to varying degrees, and most of the proteins were involved in the reaction at least to some extent. A distinct group of proteins from each subunit were, however, reproducibly cross-linked in high yield.The formaldehyde-treated subunits were subjected to a controlled hydrolysis with ribonuclease TI, and RNA fragments of known nucleotide sequence containing the cross-linked proteins were isolated. Despite the reversibility of the cross-linking reaction, sufficient cross-linked protein survived the experiment to enable an analysis to be made of proteins associated with the various RNA fragments.In the 50-S subunit, proteins L27 and L32 were found to be cross-linked to the 18-S RNA fragment which arises from the 3' end of 23-S RNA. L2, L6 and L16 were also found in this fragment in small amounts. In contrast, L13 appeared to be linked to both the 18-S RNA and the 13-S RNA fragment from the 5' end of 23-S RNA. In the 30-S subunit, proteins S3, S5, S9 (ll), S 13, and to some extent S4, S7, S 12 and S 18, were found associated with an RNA region comprising approximately 900 nucleotides at the 5' end of 16-S RNA. S4, S9 (ll), S13, and to a lesser extent S 7 and S14, were found in an RNA region comprising about 450 nucleotides near the 3' end of 16-S RNA, but lacking the 3'-terminal 150 nucleotides. Protein S21 was cross-linked to 16-S RNA, but was not found in either of the two RNA regions above.Two different biochemical approaches are currently being used to investigate RNA-protein interactions in the Escherichia coli ribosome. The first of these involves the study of complexes between isolated proteins and ribosomal RNA or fragments of ribosomal , and this method is basically intended to discover those regions of the RNA which contain stable 'binding sites' for individual proteins. The second approach involves the isolation of ribonucleoprotein fragments from ribosomal subunits, the purpose here being to investigate topographical relationships 'between proteins or groups

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Section: Analysis Of 50-s Subunit Rna Regionsmentioning

confidence: 99%

The Use of Formaldehyde in RNA‐Protein Cross‐linking Studies with Ribosomal Subunits from Escherichia coli

Möller

Rinke

Ross

et al. 1977

European Journal of Biochemistry

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“…Other workers [27] have found that four proteins, namely L2, L6, L18 and L25, are required to attach 5-S RNA to the 23-S RNA in a partial reconstitution system, and that the 3'-proximal half of the 23-S RNA is involved in the formation of this complex. Both L2 and L6 are 23-S-RNA-binding proteins [16,28], but neither was found in our fragment. The point of particular importance is that the fragment containing L5, L18 and L25 must clearly contain at least some 23-S RNA sequences, and yet none of the three proteins in the fragment is a 23-S RNA-binding protein [16,28].…”

Section: Discussionmentioning

confidence: 59%

“…2. Radioactivity of each protein in every gel slice is shown, after protein [16] which attaches to the 3'-proximal half of the 23-S RNA (P. Spierer, R. A. Zimmermann, and G. A. Mackie, unpublished results), and it is also involved at the 30-S-50-S sub-particle interface [18]. On the other hand, little is known at present about L9.…”

Section: Discussionmentioning

confidence: 99%

Two Specific Ribonucleoprotein Fragments from the 50-S Sub-Particle of Escherichia coli Ribosomes

Newton

Brimacombe

1974

Eur J Biochem

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50-S ribosomal particles from Escherichia coli were hydrolysed with ribonuclease TI, and the ribonucleoprotein fragments produced were fractionated on polyacrylamide gel. The proteins contained in these fragments were analysed on gels containing the detergent sarkosyl (N-laurylsarcosine), using the technique already published. Two-dimensional gel electrophoresis was used for the final identification of the proteins.Two very specific fragments were obtained, the smaller containing only proteins L1 and L9, and the larger containing only L5, L18 and L25. The latter proteins have been shown by other authors to be involved in binding to 5-S RNA, but in this case the fragment contained more RNA than can be accounted for by 5-S RNA alone. This offers the possibility of determining the regions of 23-S RNA associated both with these two groups of proteins and with 5-S RNA. Both fragments are discussed in terms of a possible oligonucleotide analysis of their RNA moieties.It has been shown that under suitably controlled conditions 30-S ribosomal sub-particles from Escherichia coli can be digested with nuclease to yield specific fragments of ribonucleoprotein [l, 21. Such fragments have proved useful in several ways. Analysis of their protein content provides information as to the grouping of individual proteins within the ribosome [2], while analysis of the constituent RNA sequences can show which regions of the ribosomal RNA are associated with a particular group of proteins [3]. Further, such fragments have been used in more functionally oriented studies, such as the binding of streptomycin [l].A similar fragmentation approach has been used by other authors on the 50-S sub-particle. Allet and Spahr [4] isolated two large ribonucleoprotein fragments which showed some specificity in their protein content. Kagawa et al. [5] have reported a series of small fragments all from the 50-S sub-particle, but unfortunately were not able to described their proteins in the accepted nomenclature [6]. In this paper we describe the isolation of two small very specific ribo-The preceding paper in this series is by Pongs and Nierhaus (Mol. Gen. Genet., in press).Enzyme. Ribonuclease T, (EC 3.1.4.8).Definition. An A,,, unit is the quantity of material contained in 1 ml of a solution which has an absorbance of 1 at 260 nm, when measured in a l-cm path-length cell.-~ nucleoprotein fragments from the Escherichia coli 50-S ribosome, using the methodology we have already described for the 30-S sub-particle [2,7], extended to include two-dimensional gel electrophoresis [8] for the final identification of the proteins involved. Preliminary experiments have shown that RNA fragments of the expected order of size can be isolated from these RNA . protein complexes, suggesting that both fragments could be usefully subjected to oligonucleotide analysis. MATERIALS AND METHODS Preparation of RibosomesNH,Cl-washed ribosomal sub-particles were prepared as described previously [7], with the exception that E. coli strain A19 was used instead of MRE 600. Rib...

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“…This approach has been successfully applied, so far, to proteins S4, S8, S15 and S20 on 16-S RNA [l], proteins L18 and L25 on 5-S RNA [2] and to proteins L20, L23 and L24 on 2 3 4 RNA [3,4]. In the present work, we describe the isolation and characterisation of a ribonucleoprotein containing protein L1.Protein L1 is one of the largest ribosomal proteins and binds directly to 23-S RNA of Escherichia coli [5]. It has been located in the subunit interface region of the 50-S subunit by antibody binding [6].…”

mentioning

confidence: 97%

“…Protein L1 is one of the largest ribosomal proteins and binds directly to 23-S RNA of Escherichia coli [5]. It has been located in the subunit interface region of the 50-S subunit by antibody binding [6].…”

mentioning

confidence: 99%

The Binding Site of Protein L1 on 23‐S Ribosomal RNA of Escherichia coli

Sloof¹,

Garrett²,

Krol³

et al. 1976

European Journal of Biochemistry

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Ribonucleoproteins were prepared by ribonuclease digestion of a reconstituted complex of ribosomal protein L 1 and 23-S RNA from Escherichia coli. Three main ribonucleoproteins were identified. The largest was only obtained in an impure state at low ribonuclease concentrations, whereas the two smaller ones, which were difficult to separate from one another electrophoretically, were stable over a range of enzyme concentrations.The two smaller ribonucleoproteins yielded a total of 13 RNA subfragments that were judged to be homogeneous electrophoretically. The latter were characterized for molecular weight and the subfragment composition of each of these ribonucleoproteins was established. Furthermore, the subfragments were shown to be maintained together in each ribonucleoprotein by RNA-RNA interactions.The primary and specific binding site of protein L 1 was localized on one continuous RNA subfragment of about 110 nucleotides in length by two newly developed binding methods.One approach to elucidating the ribosomal structure is to isolate specific ribosomal fragments that are sufficiently small for detailed structural analyses using physical-chemical and physical methods. To this end, ribonucleoproteins have been isolated containing a single protein and its RNA binding region by ribonuclease digestion of protein . RNA com-plexes. This approach has been successfully applied, so far, to proteins S4, S8, S15 and S20 on 16-S RNA [l], proteins L18 and L25 on 5-S RNA [2] and to proteins L20, L23 and L24 on 2 3 4 RNA [3,4]. In the present work, we describe the isolation and characterisation of a ribonucleoprotein containing protein L1.Protein L1 is one of the largest ribosomal proteins and binds directly to 23-S RNA of Escherichia coli [5]. It has been located in the subunit interface region of the 50-S subunit by antibody binding [6]. Moreover, it is essential for the association of ribosomal subunits (together with either L11 or L16), and for the formation of the 70-S ribosomal initiation complex [7].Here, we demonstrate that three specific ribonucleoproteins, containing L1 and differing amounts ~~ Enzyme. T1 RNase (EC 3.1.4.8).of RNA, can be produced by TI ribonuclease digestion of a complex of L1 and 23-S RNA. The structural homogeneity of the RNA component of these ribonucleoproteins was investigated for (a) different degrees of ribonuclease digestion and (b) different magnesium concentrations during digestion. The optimal conditions for preparing each ribonucleoprotein were established and the RNA subfragments contained in them were identified and characterised for molecular weight. Furthermore, two new methods were developed for determining the smallest RNA region required for specific protein L1 binding. Nucleotide sequence studies on these RNA components are given in two subsequent papers 91. MATERIALS AND METHODS Preparation of 23-S R N A , Protein L1 and L l .23-S-RNA Complex32P-labelled and unlabelled 23-S RNA were extracted from 50-S subunits of E. coli (strains A19 and MRE 600) by three successive phenol/d...

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Binding of 50 S ribosomal subunit proteins to 23 S RNA of Escherichia coli

Cited by 61 publications

References 13 publications

The Use of Formaldehyde in RNA‐Protein Cross‐linking Studies with Ribosomal Subunits from Escherichia coli

The Use of Formaldehyde in RNA‐Protein Cross‐linking Studies with Ribosomal Subunits from Escherichia coli

Two Specific Ribonucleoprotein Fragments from the 50-S Sub-Particle of Escherichia coli Ribosomes

The Binding Site of Protein L1 on 23‐S Ribosomal RNA of Escherichia coli

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