Ribosome-bound eukaryotic elongation factor 2 protects 5 S rRNA from modification.

Holmberg, Lovisa; Melander, Yvette; Nygård, Odd

doi:10.1016/s0021-9258(19)36698-0

Cited by 20 publications

(9 citation statements)

References 35 publications

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“…Chemical cross-linking performed in prokaryotes shows that 23S rRNA is located in close proximity to EF-G (Skold, 1983), whereas attempts to cross-link eEF-2 to 18S or 28S rRNA in eukaryotes using the same bifunctional reagent have been unsuccessful (Nyg&rd & . However, eEF-2 can be cross-linked to 5S rRNA, and ribosome-bound factor also gives specific chemical and enzymatic footprints on 5S rRNA (Holmberg et al, 1992).…”

Section: Discussionmentioning

confidence: 99%

“…(e) Modification of rRNA. Modification of rRNA was as previously described (Holmberg et al, 1992). The modifying reagent was added to the samples containing assembled control 80S ribosomes or 80S ribosomes in a 1:1 complex with eEF-2*Guo/>/>[CH2]P. The reagents DMS and CMCT were added at a final concentration of 20 or 90 /<M for DMS and 20 or 100 mM for CMCT.…”

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confidence: 99%

“…The modifying reagent was added to the samples containing assembled control 80S ribosomes or 80S ribosomes in a 1:1 complex with eEF-2*Guo/>/>[CH2]P. The reagents DMS and CMCT were added at a final concentration of 20 or 90 /<M for DMS and 20 or 100 mM for CMCT. Enzymatic cleavage with MNase was performed at 0.2 or 0.9 /<M of the enzyme (Holmberg et al, 1992). Control samples were incubated without modifying reagents but were otherwise treated identically.…”

mentioning

confidence: 99%

“…End-labeling of the cDNA primers, primer extension, dideoxy sequencing, gel electrophoresis, and autoradiography were as previously described (Holmberg et al, 1992(Holmberg et al, ,1994a. The autoradiograms were quantified using a computerassisted image analysis system.…”

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confidence: 99%

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Interaction Sites of Ribosome Bound Eukaryotic Elongation Factor 2 in 18S and 28S rRNA

Holmberg

Nygård²

1994

Biochemistry

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The involvement of ribosomal RNA in the binding of eukaryotic elongation factor eEF-2 to the ribosome was investigated. eEF-2 was complexed to empty reassociated 80S ribosomes in the presence of the nonhydrolyzable GTP analogue GuoPP[CH2]P. The formed complex was treated with dimethyl sulfate, 1-cyclohexyl-3-(2-morpholinoethyl)carbodiimide metho-p-toluenesulfonate, and micrococcus nuclease to allow specific modification at single-stranded regions of the rRNAs. The sites of modification were localized by primer extension using complementary deoxynucleotide primers and reverse transcriptase. The modification pattern was compared to that obtained from 80S ribosomes lacking bound eEF-2. Binding of the factor to the ribosome resulted in the protection of specific sites in both 18S and 28S rRNA, while the reactivity of 5.8S rRNA was unchanged. In 18S rRNA, the affected nucleotides were localized to the 5'- and 3'-domains, and in 28S rRNA the protected nucleotides were seen in domains II, IV, and V. The alpha-sarcin/ricin loop in domain VI of 28S rRNA was inaccessible for chemical modification even in the absence of bound eEF-2. However, the bound factor protected A4256, located in the alpha-sarcin/ricin loop, from ricin-induced depurination.

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Section: Discussionmentioning

confidence: 99%

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Interaction Sites of Ribosome Bound Eukaryotic Elongation Factor 2 in 18S and 28S rRNA

Holmberg

Nygård²

1994

Biochemistry

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“…Identification of Modification Sites. The positions of the modified bases and/or cleavage sites were identified using primer extension as previously described (35). The primers are listed in Table 1.…”

Section: Methodsmentioning

confidence: 99%

Probing the Secondary Structure of Expansion Segment ES6 in 18S Ribosomal RNA

Alkemar

Nygård

2006

Biochemistry

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Expansion segment ES6 in 18S ribosomal RNA is, unlike many other expansion segments, present in all eukaryotes. The available data suggest that ES6 is located on the surface of the small ribosomal subunit. Here we have analyzed the secondary structure of the complete ES6 sequence in intact ribosomes from three eukaryotes, wheat, yeast, and mouse, representing different eukaryotic kingdoms. The availability of the ES6 sequence for modification and cleavage by structure sensitive chemicals and enzymatic reagents was analyzed by primer extension and gel electrophoresis on an ABI 377 automated DNA sequencer. The experimental results were used to restrict the number of possible secondary structure models of ES6 generated by the folding software MFOLD. The modification data obtained from the three experimental organisms were very similar despite the sequence variation. Consequently, similar secondary structure models were obtained for the ES6 sequence in wheat, yeast, and mouse ribosomes. A comparison of sequence data from more than 6000 eukaryotes showed that similar structural elements could also be formed in other organisms. The comparative analysis also showed that the extent of compensatory base changes in the suggested helices was low. The in situ structure analysis was complemented by a secondary structure analysis of wheat ES6 transcribed and folded in vitro. The obtained modification data indicate that the secondary structure of the in vitro transcribed sequence differs from that observed in the intact ribosome. These results suggest that chaperones, ribosomal proteins, and/or tertiary rRNA interactions could be involved in the in vivo folding of ES6.

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Effect of oxidizing agents and haemin on the phosphorylation of eukaryotic elongation factor 2 in rabbit reticulocyte lysates

Nilsson

Nygård

1995

Biochimica et Biophysica Acta (BBA) - Gene Structure and Expres

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Ribosome-bound eukaryotic elongation factor 2 protects 5 S rRNA from modification.

Cited by 20 publications

References 35 publications

Interaction Sites of Ribosome Bound Eukaryotic Elongation Factor 2 in 18S and 28S rRNA

Interaction Sites of Ribosome Bound Eukaryotic Elongation Factor 2 in 18S and 28S rRNA

Probing the Secondary Structure of Expansion Segment ES6 in 18S Ribosomal RNA

Effect of oxidizing agents and haemin on the phosphorylation of eukaryotic elongation factor 2 in rabbit reticulocyte lysates

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