Interactions of the N-terminal Domain of Ribosomal Protein L11 with Thiostrepton and rRNA

Sarae L Bausch,Ekaterina Poliakova,David E Draper

doi:10.1074/jbc.m504182200

Sarae L Bausch, Ekaterina Poliakova + Show 1 more

Open Access

https://doi.org/10.1074/jbc.m504182200

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Abstract

Ribosomal protein L11 has two domains: the C-terminal domain (L11-C76) binds rRNA, whereas the N-terminal domain (L11-NTD) may variously interact with elongation factor G, the antibiotic thiostrepton, and rRNA. To begin to quantitate these interactions, L11 from Bacillus stearothermophilus has been overexpressed and its properties compared with those of L11-C76 alone in a fluorescence assay for protein-rRNA binding. The assay relies on 2'-amino-butyryl-pyrene-uridine incorporated in a 58-nucleotide rRNA fragment, which gives approximately 15-fold enhancement when L11 or L11-C76 is bound. Although the pyrene tag weakens protein binding, unbiased protein-RNA association constants were obtained in competition experiments with untagged RNA. It was found that (i) intact B. stearothermophilus L11 binds rRNA with K approximately 1.2 x 10(9) m(-1) in buffers with 0.2 m KCl, about 100-fold tighter than Escherichia coli L11; (ii) the N-terminal domain makes a small, salt-dependent contribution to the overall L11-RNA binding affinity (approximately 8-fold enhancement at 0.2 m KCl), (iii) L11 stimulates thiostrepton binding by 2.3 +/- 0.6 x 10(3)-fold, predicting an overall thiostrepton affinity for the ribosome of approximately 10(9) m(-1), and (iv) the yeast homolog of L11 shows no stimulation of thiostrepton binding. The latter observation resolves the question of why eukaryotes are insensitive to the antibiotic. These measurements also show that it is plausible for thiostrepton to compete directly with EF-G.GDP for binding to the L11-RNA complex, and provide a quantitative basis for further studies of L11 function and thiostrepton mechanism.

Highlights

It was found that (i) intact B. stearothermophilus L11 binds rRNA with K Ϸ 1.2 ؋ 109 M؊1 in buffers with 0.2 M KCl, about 100-fold tighter than Escherichia coli L11; (ii) the N-terminal domain makes a small, salt-dependent contribution to the overall L11-RNA binding affinity (ϳ8-fold enhancement at 0.2 M KCl), (iii) L11 stimulates thiostrepton binding by 2.3 ؎ 0.6 ؋ 103-fold, predicting an overall thiostrepton affinity for the ribosome of ϳ109 M؊1, and (iv) the yeast homolog of L11 shows no stimulation of thiostrepton binding
1 The abbreviations used are: L11 BD RNA, nucleotides 1051–1108 of the 23 S rRNA (E. coli numbering) that constitute the minimal binding domain for protein L11; L11-NTD, N-terminal domain of L11; L11-C76, C-terminal RNA binding domain of L11; THS, thiostrepton; EF-G, elongation factor G; DTT, dithiothreitol; MOPS, 4-morpholinepropanesulfonic acid; py-U1082; RNA, L11 BD RNA modified at position U1082 with 2Ј-amino-butyryl-pyrene-uridine
The py-U1082 location of the tag used here was chosen with the anticipation that its proximity to bound L11 would induce a change in fluorescence intensity

Summary

The abbreviations used are

L11 BD RNA, nucleotides 1051–1108 of the 23 S rRNA (E. coli numbering) that constitute the minimal binding domain for protein L11; L11-NTD, N-terminal domain of L11; L11-C76, C-terminal RNA binding domain of L11; THS, thiostrepton; EF-G, elongation factor G; DTT, dithiothreitol; MOPS, 4-morpholinepropanesulfonic acid; py-U1082; RNA, L11 BD RNA modified at position U1082 with 2Ј-amino-butyryl-pyrene-uridine. We describe the development of a fluorescence assay that allows accurate measurement of RNA affinities for L11 or L11-C76 under a wide variety of solution conditions and in the presence or absence of thiostrepton Using this assay, we show that the bacterial L11-NTD contributes a small, salt-dependent free energy to L11-rRNA binding affinities, and enhances thiostrepton-rRNA interactions by factors of more than 103. We show that the bacterial L11-NTD contributes a small, salt-dependent free energy to L11-rRNA binding affinities, and enhances thiostrepton-rRNA interactions by factors of more than 103 This cooperative interaction does not take place with a eukaryotic L11 homolog, which accounts for the resistance of eukaryotic ribosomes to the drug

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DISCUSSION