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

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

Sarae L. Bausch; Ekaterina Poliakova; David E. Draper

2005

Scholarcy highlights

  • It was found that 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; the N-terminal domain makes a small, salt-dependent contribution to the overall L11-RNA binding affinity, L11 stimulates thiostrepton binding by 2.3 ؎ 0.6 ‫ ؋‬103-fold, predicting an overall thiostrepton affinity for the ribosome of ϳ109 M؊1, and the yeast homolog of L11 shows no stimulation of thiostrepton binding
  • Cloning and Purification of L11—The L11 gene from B. stearothermophilus was previously cloned and expressed based on a sequence of ribosome-purified protein reported by the Wittman-Liebold laboratory
  • An likely explanation is that the pyrene fluorescence is quenched by stacking with bases in a partially unfolded form of the RNA. This would account for both tertiary structure destabilization and the fluorescence enhancement induced by ligands
  • Similar behavior of pyrene tags has been seen in other nucleic acid systems
  • Ϳ100-fold differences in the affinities of E. coli and B. stearothermophilus ribosomal protein-rRNA complexes in two other cases, S4 and L3.4 A systematic study of ribosomal protein S8 homologs from meso, thermo, and hyperthermophilic sources found a 100-fold range in rRNA binding affinities that correlated with growth temperature
  • These are small contributions compared with the overall Ϫ12.4 kcal/mol binding free energy for L11-C76 binding rRNA in buffer with 0.2 M KCl. Such weak protein-RNA interactions may take place because the N-terminal domain is constrained near the rRNA surface

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