The identification of the RNA binding site for a 50 s ribosomal protein by a new technique

Branlant, Christiane; Krol, Alain; Sri-Widada, Johanes; Fellner, Peter

doi:10.1016/0014-5793(73)80301-1

Cited by 45 publications

(31 citation statements)

References 21 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…These results are schematized in fig.4, together with the previous findings of Branlant et al [2,3] and Spierer et al [4]. There is a good agreement between this work and the report of Branlant et al [3], even though these authors used different techniques for the localization of protein binding sites on the 23 S RNA.…”

Section: Discussionsupporting

confidence: 85%

See 1 more Smart Citation

RNA—protein interactions in the ribosome. Binding of proteins L1, L3, L6, L13 and L23 to specific fragments of the 23S RNA

Spierer¹,

Zimmermann²

1976

FEBS Letters

View full text Add to dashboard Cite

Ten of the 33 proteins found in the 50 S subunit of the Escherichia coli ribosome associate independently and specifically to the 23 S ribosomal RNA [l]. The location of the binding sites for these proteins has been studied in two different ways. Branlant et al. [2,3] have isolated ribonucleoprotein particles resulting from extensive nuclease digestion of complexes between proteins Ll, L20, L23, L24 and the 23 S RNA. Sequence analysis of the RNA regions protected from digestion by the proteins enabled these authors to locate the binding sites on the 23 S RNA molecule. On the other hand, Spierer et al. [4] have tested the association of each of the ten binding proteins with two segments of the 23 S RNA produced by limited ribonuclease digestion of the 50 S subunit. One segment sediments at 13 S, covers 40% of the 23 S RNA sequence, and derives from the 5' end of the molecule. It contains the specific binding sites for proteins LA, L20 and L24. The other segment, which sediments at 18 S, encompasses about 60% of the 23 S RNA sequence and arises from the 3' end of the molecule. It contains the specific binding sites for proteins Ll, L2, L3, L6, L13, L16 and L23. We report here the preparation of fragments of the 18 S RNA segment, and the distribution of binding sites for proteins Ll, L.3, L6, L13 and L23 on these fragments

show abstract

Section: Discussionsupporting

confidence: 85%

“…Branlant et al [2,3] have isolated ribonucleoprotein particles resulting from extensive nuclease digestion of complexes between proteins Ll, L20, L23, L24 and the 23 S RNA. Sequence analysis of the RNA regions protected from digestion by the proteins enabled these authors to locate the binding sites on the 23 S RNA molecule.…”

Section: Introductionmentioning

confidence: 99%

RNA—protein interactions in the ribosome. Binding of proteins L1, L3, L6, L13 and L23 to specific fragments of the 23S RNA

Spierer¹,

Zimmermann²

1976

FEBS Letters

View full text Add to dashboard Cite

show abstract

“…The RNA fragments were analysed by two methods. (a) The RNA was eluted onto Whatman DEAE 81 paper, electrophoretically (23). It was then eluted from the paper with triethylamine carbonate, pH 10, and dried.…”

Section: Methodsmentioning

confidence: 99%

“…(b) The gel piece was soaked for 3 hr in 8 M urea in water to dissociate base-paired regions, and then set on a composite 15 to 20% polyacrylamide gel containing 0.09 M Tris-borate, pH 8.3, 2.5 mM EDTA and 7 M urea; a short upper layer contained 0.1% dodecylsulphate (23). The RNA subfragments, resolved by the electrophoresis, were eluted onto DEAE 81 paper electrophoretically (23) and further analysed as above.…”

Section: Methodsmentioning

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

View full text Add to dashboard Cite

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.

show abstract

“…These were pooled as indicated for polyacrylamide gel analysis (see Fig. 10) 11. Poljucrylm?ide disc gel electrophoresis of30-S ribosomulproteins clzronioft~~ruplze~/ on tRNA-ugarose.…”

Section: Ajjin Ity Chroma Togruphy ~F E Coli 30-s Ribosomal Proteinsmentioning

confidence: 99%

Binding of Ribosomal Proteins to RNA Covalently Coupled to Agarose

Burrell

Horowitz

1977

Eur J Biochem

View full text Add to dashboard Cite

An affinity chromatographic procedure, using ribosomal or tRNA covalently coupled to agarose through an adipic acid dihydrazide spacer, has been developed to isolate and identify the Escherichia coli ribosomal proteins which specifically bind to these RNA molecules. Transfer RNA, 5-S, 16-S and 23-S ribosomal RNA were readily immobilized on agarose-dihydrazide: the maximum amount coupled decreasing with increasing molecular size. Specific binding of ribosomal proteins to RNA was observed in buffer containing 0.3 M KCI and 0.02 M MgC12. Bound proteins were eluted with a high-salt, 2 M KCI, buffer containing 0.005 M EDTA. Two ribosomal proteins, identified as L18 and L25 by two-dimensional gel electrophoresis, bound tightly to E. cob 5-S RNA;' small amounts of a third protein, L5, also were bound. These proteins did not bind to immobilized tRNA nor did 30-S ribosomal proteins bind to 5-S RNA. Denatured 5-S RNA bound less L l 8 and L25 than the native species. Several 30-S and 50-S ribosomal proteins bind to immobilized tRNA. The major 5 0 3 subunit proteins tightly bound to tRNA were identified as L3, L4, L5, L7 and L8/L9. Smaller amounts of proteins L1, L2, L11, L16 and L21 were also observed. Proteins L2 and L16 were retarded by agarose-bound tRNA. Protein S3 was the major 30-S subunit protein bound to tRNA. Lesser quantities of proteins S6, S9, S13 and S18 also interacted with tRNA.Studies of ribosomal protein -RNA interactions can give important information on the role of individual components in the structure and function of the ribosome. The discovery that the procaryotic ribosome can be self-assembled in vitvo [1,2] and the continuing elucidation of the highly intricate reaction sequence of protein biosynthesis, involving reversible associations of the ribosome with various exogenous molecules such as messenger RNA, transfer RNA, and nucleotides, suggest u pviovi that protein -protein and protein -nucleic acid interactions are essential in these complex macromolecular processes. Support for this supposition has come from several experimental approaches. Reconstitution experiments using purified ribosomal constituents have permitted a tentative identification of the ribosomal proteins that selectively bind to 5-S RNA [3-51, 16-S RNA [6-lo], and 2 3 4 RNA [ l l -141 and thus presumably are those vital to the structural integrity of the ribosome. Moreover, an impressive amount of data delineating functional sites on the ribosome has accumulated from recent investigations. utilizing such approaches From a correlation of these data one can infer that a surprisingly large number of ribosomal proteins from both subunits may be involved in interactions with extraribosomal RNA and protein factors during translation. The results, however, have not allowed an unambiguous identification of the elements comprising ribosomal active sites, perhaps owing to the indirect nature of these probes and to difficulties in interpretation caused by the high degree of cooperativity inherent in situ.In an attempt to develop an alterna...

show abstract

The identification of the RNA binding site for a 50 s ribosomal protein by a new technique

Cited by 45 publications

References 21 publications

RNA—protein interactions in the ribosome. Binding of proteins L1, L3, L6, L13 and L23 to specific fragments of the 23S RNA

RNA—protein interactions in the ribosome. Binding of proteins L1, L3, L6, L13 and L23 to specific fragments of the 23S RNA

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

Binding of Ribosomal Proteins to RNA Covalently Coupled to Agarose

Contact Info

Product

Resources

About