To prime ribosomes with poly(U), reaction mix (100 ml) in binding buffer (80 mM Tris-HCl, pH 7.4, 100 mM NaCl, 15 mM Mg(CH 3 COOH) 2 , 6mM b-mercaptoethanol) containing 55 pmol ribosomes and 50 mg poly(U), was incubated for 20 min at 30 C. Next, for P-site complex, 200 pmol of Ac-Phe-tRNA Phe was added. For structure probing of ribosomes with occupied A sites, ribosomal P sites were blocked with 4Â excess of deacylated tRNA Phe and Phe-tRNA Phe (200 pmol), GTP (0.5 mM) and 5 ml of crude elongation factor mix were added. Reaction mixes were incubated for 20 min at 30 C. Reactions were divided into two parts of 50 ml each (control and modification tubes) and 75 ml of binding buffer was added to each tube. 25 ml of 1M7 (130 mM in DMSO) was added to modification tubes. Control samples contained 25 ml of DMSO. After incubating for 85 min at 30 C ribosomes were precipitated with 450 ml ethanol. Ribosomal RNA was extracted using RNAqueous kit (Ambion). Pellets were dissolved in 100 ml of RNAqueous Lysis Buffer and processed according manufactures instructions. RNA was eluted in 50 ml volume and concentration adjusted to 1 mg/ml with elution buffer. Reverse transcriptase (RT) primer extension analyses of modified RNAs were performed as described (34) . The site-specificity of charged tRNA binding was confirmed using the puromycin reaction (35) .