To footprint, binding of mRNAs was performed by incubating 70 S ribosome (in the range of 50-750 pmol according to the mRNA tested) with mRNA (10-50 pmol) in 50 ml reaction buffer A (20 mM MgCl 2 , 150 mM NH 4 Cl, 80 mM potassium cacodylate, pH 7.2) at 37 C for 10 min. A first tRNA (320 pmol) was added to fill the P site by incubation at 37 C for 10 min. A second tRNA (320 pmol) was added to fill the A site by incubation at 37 C for 10 min. mRNA-tRNA-70 S complexes were then purified by ultrafiltration (MICROCON YM-100 100 000 Da, Fisher scientific LABOSI). The ternary complex was diluted in 250 ml of buffer A and distributed into 50 ml aliquots (2 pmol mRNA). Chemical probing (38) was performed by addition of 2, 4 or 8 ml dimethyl sulfate (DMS; 1:10 dilution in 95% ethanol), 2, 4 or 8 ml kethoxal (KE; 19 mg/ml in H 2 O) on 50 ml aliquot, followed by incubation at 37 C for 10 min. All modification reactions were stopped by addition of 150 ml 95% ethanol and 5 ml 3 M sodium acetate followed by rapid mixing. KE-modified samples were adjusted to 25 mM potassium borate (pH 7.0). The pellets were resuspended in 200 ml of 0.3 M sodium acetate, 2.5 mM EDTA and 0.5% SDS (with addition of 25 mM potassium borate for KE samples), extracted three times with phenol, twice with chloroform and resuspended in 10 ml H 2 O (for DMS samples) or in 10 ml 25 mM potassium borate (for KE samples). Primer extension reactions were performed as described (38) .