Interactions of the N-terminal domain of ribosomal protein L11 with thiostrepton and rRNA.
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.
Department of Chemistry, Johns Hopkins University, Baltimore, Maryland 21210, USA.
Sarae L
SL
Bausch
Ekaterina
E
Poliakova
David E
DE
Draper
D000900:Anti-Bacterial Agents / Q000494:pharmacology*; D003001:Cloning, Molecular; D005453:Fluorescence; D001411:Geobacillus stearothermophilus / Q000737:chemistry*; D012335:RNA, Ribosomal / Q000737:chemistry* / Q000378:metabolism; D012269:Ribosomal Proteins / Q000737:chemistry* / Q000378:metabolism; D013883:Thiostrepton / Q000378:metabolism / Q000494:pharmacology*
D000900:Anti-Bacterial Agents; D012335:RNA, Ribosomal; D012269:Ribosomal Proteins; C027624:ribosomal protein L11; D013883:Thiostrepton