Random exchange of ribosomal proteins in EDTA sub‐particles

Newton, I; Rinke, Jutta; Brimacombe, Richard

doi:10.1016/0014-5793(75)80890-8

Cited by 35 publications

(13 citation statements)

References 20 publications

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“…Chelating agents such as EDTA have been successfully employed to partially unfold a number of RNPs including ribosomal subunits, RNase P, small cytoplasmic RNPs and SRP, which were subsequently disassembled into protein and RNA components by column chromatography (Blobel 1971;Spirin 1974;Newton et al 1975;Guthrie and Atchison 1980;Mukherjee and Sarkar 1981;Walter and Blobel 1983). To address the effect of EDTA on reconstitution of telomerase activity, we first treated partially purified active telomerase fractions with Ca^"^ and MNase to remove endogenous telomerase RNA.…”

Section: Reconstitution Of Telomerase Activity After Mnase Digestionmentioning

confidence: 99%

“…Studies on ribosomes suggest that EDTA does not cause the disassembly of ribosomes into RNA and protein, because after treatment the RNA and protein cosediment on sucrose gradients. Rather EDTA alters the specificity of the protein-RNA interaction such that proteins are free to move from one RNA strand to another (Newton et al 1975). Thus, the addition of EDTA during reconstitution of telomerase activity may stimulate the interaction between inactivated MNase-treated telomerase and exogenously added synthetic telomerase RNA.…”

Section: Reconstitution Of Telomerase Activitymentioning

confidence: 99%

See 1 more Smart Citation

Functional reconstitution of wild-type and mutant Tetrahymena telomerase.

Autexier¹,

Greider²

1994

Genes Dev.

100

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Telomerase is a ribonucleoprotein that catalyzes telomere elongation in vitro and in vivo. The 159-nucleotide RNA component of Tetrahymena telomerase contains the sequence 5'-CAACCCCAA-3' ("template region"), which serves as a template for the addition of the sequence d(TTGGGG)" to Tetrahymena telomeres. To dissect the Tetrahymena telomerase enzyme mechanism, we developed a functional in vitro reconstitution assay. After removal of the essential telomerase RNA by micrococcal nuclease digestion of partially purified telomerase, the addition of in vitro-transcribed telomerase RNA reconstituted telomerase activity. The reconstituted activity was processive and showed the same primer specificities as native telomerase. Mutants in the RNA template region were tested in reconstitution assays to determine the role of the residues in this region in primer recognition and elongation. Two template mutants, encoding the sequences 5'-UAACCCCAA-3' and 5'-UAACCCUAA-3', specified the incorporation of dATP into the sequence d(TTAGGG). Telomerase reconstituted with a template mutant encoding the sequence 5'-CAACCCUAA-3' did not specify dATP incorporation and elongation by this mutant was not terminated by the addition of ddATP. In addition, a template mutant encoding the sequence 5'-CGGCCCCAA-3' specified the incorporation of ddCTP but not ddTTP while a mutant encoding the sequence 5'-CAACCCCGG-3' specified the incorporation of ddTTP but not ddCTP. These data suggest that only the most 5' six residues of the template region dictate the addition of telomeric repeats.

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Section: Reconstitution Of Telomerase Activity After Mnase Digestionmentioning

confidence: 99%

Section: Reconstitution Of Telomerase Activitymentioning

confidence: 99%

Functional reconstitution of wild-type and mutant Tetrahymena telomerase.

Autexier¹,

Greider²

1994

Genes Dev.

100

View full text Add to dashboard Cite

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“…2, fractions 25 -28) shows little specificity, due either to contamination with further breakdown products from fragment I1 (cf: Fig. 1) or to a partial randomisation of the proteins among the RNA fragments under these hydrolysis conditions (~f : [22]). It is also clear that the deoxycholate washes some proteins off, as evidenced by the total absence of S9 from the fragment 111 region (Fig, 2).…”

Section: Anulysis Of a Small Rihonucleoprotein Fragment Containing Prmentioning

confidence: 99%

Studies on the Environment of Protein S7 within the 30‐S Subunit of Escherichia coli Ribosomes

Rinks

Yûki

Brimacombe

1976

European Journal of Biochemistry

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Analyses are described of three types of ribosomal fragment, all derived from the well-characterized ribonucleoprotein species consisting of proteins S7, S9, S 10, S 14 and S 19, together with RNA from sections 0'-D-E'-K-P-P'-E-A of the 16-S sequence.1. When 30-S subunits were hydrolysed with ribonuclease T, in the presence of deoxycholate in addition to the components previously described, a fragment could be isolated which contained only proteins S7 and S19, together with minor amounts of S13 or S14. Oligonucleotide analysis of this fragment showed that it contained RNA from sections 0'-E' and P-A of the 16-S RNA, but that section K (from the middle of this area) was missing.2. It has previously been shown that when 30-S subunits are irradiated with ultraviolet light, protein S7 is the primary target of cross-linking of protein to RNA. By making use of this reaction, ribonucleoprotein fragments were isolated from irradiated 30-S subunits, the unbound proteins were removed, and RNA fragments containing covalently linked protein S7 were identified. It was possible to demonstrate that the site of cross-linking lies within the 0'-A region of the 26-S RNA, and more precise experiments showed that this site almost certainly is in the P-A region.3. When the parent five-protein ribonucleoprotein fragment (above) was deproteinized under very mild conditions, RNA complexes could be isolated which consisted of non-contiguous sequences, but which migrated as a single species into a polyacrylamide gel. Analysis of one of these complexes showed that it contained sequences from sections 0'-E' and P-A, but that section K was missing (cf: first paragraph above). This demonstrated that these two separate regions of RNA interact within the 30-S subunit, independently of the presence of protein.

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“…The deprivation of Mg2+ produces an unfolding of the particles [21,22] and removes from the large subunit a ribonucleoprotein particle of about 5 S [25] containing the 5-S RNA and a 35000-M, ribosomal protein. We observe that the deprivation of Mg2 alone brings about a spontaneous and complete loss of three ribosomal proteins SPO, LPI, LP3 ( Table 1).…”

Section: Discussionmentioning

confidence: 99%

“…As yet, no direct comparison between the ribosomal proteins belonging to these four groups has been made. Here, we report on such a comparative study, in which two methods for dissociating ribosomes have been used concomitantly, in order to gain more insight about the ribosomal structure; one of them (EDTA) produces extensive conformational change [21,22], the other (puromycin) leaves the subunits relatively unmodified [18,23]. The ribosomal proteins extracted from the KCI-washed subunits and from the KCI supernatants have been compared.…”

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confidence: 99%

The Ribosomal Proteins of L Cells

Cazillis¹,

Houssais²

1981

European Journal of Biochemistry

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To approach the problem of the RNA-protein and protein-protein non-covalent interactions in mammalian ribosomes, the behaviour of the ribosomal proteins of L cells has been studied under various conditions. Firstly, the electrophoretic patterns of the ribosomal proteins extracted from KCI-washed subunits dissociated by EDTA or puromycin have been compared. All the ribosomal proteins of the EDTA-derived subunits are found on the puromycin-derived subunits with the exception of two (S20, S22) not observed when puromycin is used to dissociate the subunits. Three polypeptides of the puromycin-derived subunit, SPO, LP1, LP3, are stripped by the sole removal of Mg2+ when EDTA is used. Other additional polypeptides are found on the KC1-washed puromycin-derived subunits. Secondly, the dissociating effect of 0.5 M KCI on the proteins of the subunits dissociated by EDTA or puromycin has been studied. Although the EDTA-derived subunits appear much more sensitive to KC1, nevertheless 0.5 M KCI also releases ribosomal proteins from the puromycin-derived subunits. Other polypeptides (core proteins) are not susceptible to KCl split off. Thirdly, some ribosomal proteins are able to transfer from one type of subunit to the other; these proteins have been classified into three groups. Although the deprivation of Mg2+ enhances this phenomenon, seven pairs of putative transferable proteins are observed in subunits dissociated both by EDTA and puromycin. Lastly, a comparative study has been made between the proteins split off by KCI, the core proteins and the transferable proteins with the ribosomal polypeptides labelled in the absence of rRNA synthesis. A correlation has been observed between the proteins split off by KCI, the transferable proteins and the labelled proteins; however, none of the core proteins are transferable and the major part of them remain unlabelled. This study suggests that the transfer, splitting off and labelling of proteins are properties related to the topological location of the proteins in the ribosomal structure.The cytoplasmic ribosomes contain more than 70 different polypeptides [I -51. Studies of the conformation of such an RNA-protein assembly, of the location of the ribosomal polypeptides and of their function in protein synthesis, are made difficult due to the complexity of these ribonucleoprotein structures. One approach to this problem has been to study the behaviour of the ribosomal proteins under various ionic and labelling conditions. Along this line some of the eukaryotic ribosomal proteins have been described either as KCI-split-off proteins [6 -81, core proteins [9,10], proteins apparently transferable from one subunit to the other [I1 -141 or as proteins which are labelled on the cytoplasmic ribosomes when the nucleolar synthesis of the ribosomes has been suppressed [5,. As yet, no direct comparison between the ribosomal proteins belonging to these four groups has been made. Here, we report on such a comparative study, in which two methods for dissociating ribosomes have been used concomi...

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Random exchange of ribosomal proteins in EDTA sub‐particles

Cited by 35 publications

References 20 publications

Functional reconstitution of wild-type and mutant Tetrahymena telomerase.

Functional reconstitution of wild-type and mutant Tetrahymena telomerase.

Studies on the Environment of Protein S7 within the 30‐S Subunit of Escherichia coli Ribosomes

The Ribosomal Proteins of L Cells

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