Ribosomal RNAs (rRNAs) from all kingdoms contain a variety of post-transcriptional modifications and these are typically clustered in the functional centers of the ribosome. The functions of two bases in the 23S rRNA of Escherichia coli that are post-transcriptionally modified, m(5)U1939 and psi 2504, were examined by mutagenesis of the rRNA bases and by inactivation of the RumA methylase that methylates U1939. Base substitutions at U1939 had little effect on growth or the fidelity of translation, but altered the sensitivity of the ribosomes to the antibiotics fusidic acid and capreomycin. Strains lacking the RumA methylase were gradually out-competed by wild type strains ill growth competition experiments, suggesting that the m(5)U methylation improves ribosome performance. Base changes at psi 2504 had dramatic effects on growth and resistance to several peptidyltransferase inhibitor antibiotics and increased the levels of translational errors. The results link these sites of post-transcriptional modification with the ribosome's response to antibiotics and the control of translational fidelity. (C) 2010 Elsevier Inc. All rights reserved.