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ID: 24738.0, MPI für molekulare Genetik / Ribosome Research Groups
Crosslinking of 4.5S RNA to the Escherichia coli ribosome in the presence or absence of the protein Ffh
Authors:Rinke-Appel, Jutta; Osswald, Monika; von Knoblauch, Klaus; Mueller, Florian; Brimacombe, Richard; Sergiev, Petr; Avdeeva, Olga; Bogdanov, Alexey; Dontsova, Olga
Date of Publication (YYYY-MM-DD):2002-05
Title of Journal:RNA
Issue / Number:5
Start Page:612
End Page:625
Review Status:not specified
Audience:Experts Only
Abstract / Description:Radioactively labeled 4.5S RNA containing statistically distributed 4-thiouridine residues in place of normal uridine was prepared by T7 transcription. The ability of this modified 4.5S RNA to form a complex with the protein Ffh was demonstrated by a gel shift assay. The modified 4.5S RNA, with or without Ffh, was added to Escherichia coli ribosomes under various conditions, and crosslinking from the thiouridine residues was induced by irradiation at 350 nm. The crosslinked ribosomal components were analyzed by our standard procedures. Two clearly defined types of crosslinking were observed. The first was a crosslink to 23S rRNA, which was entirely dependent both on the presence of Ffh and a nascent protein chain in the 50S subunit. This crosslink was localized to nt [similar] 2828–2837 of the 23S rRNA, from position 84 of the 4.5S molecule. The second type of crosslinking, to the 30S ribosomal subunit, was independent of the presence of Ffh, and was found both with vacant 70S ribosomes or isolated 30S subunits. Here the crosslink was localized to the 3[prime prime or minute]-terminal region of the 16S rRNA, from positions 29–50 of the 4.5S RNA. Crosslinking to ribosomal protein S1 was also observed. The known crystal structure of the protein Ffh/4.5S RNA fragment complex was extrapolated by computer modeling so as to include the whole 4.5S molecule, and this was docked onto the ribosome using the crosslinking data. The results are discussed in terms of multiple functions and binding sites of the 4.5S RNA.
Free Keywords:elongation factor G; functions of 4.5S RNA; nascent peptide chain; ribosome structure; signal recognition particle
External Publication Status:published
Document Type:Article
Communicated by:Ribosome Group
Affiliations:MPI für molekulare Genetik
External Affiliations:Department of Chemistry, Moscow State University, Moscow 119899, Russia
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