Compensatory Evolution Reveals Functional Interactions between Ribosomal Proteins S12, L14 and L19 Sophie Maisnier-Patin 1 ⁎, Wilhelm Paulander 1 , Alexandra Pennhag 1 and Dan I. Andersson 1,2 1 Department of Bacteriology, Swedish Institute for Infectious Disease Control and Microbiology and Tumor Center , Karolinska Institute, S-17182 Solna, Sweden 2 Department of Medical Biochemistry and Microbiology, Uppsala University, Box 582, S-75123 Uppsala, Sweden Certain mutations in S12, a ribosomal protein involved in translation elongation rate and translation accuracy, confer resistance to the aminogly- coside streptomycin. Previously we showed in Salmonella typhimurium that the fitness cost, i.e. reduced growth rate, due to the amino acid substitution K42N in S12 could be compensated by at least 35 different mutations located in the ribosomal proteins S4, S5 and L19. Here, we have characterized in vivo the fitness, translation speed and translation accuracy of four different L19 mutants. When separated from the resistance mutation located in S12, the three different compensatory amino acid substitutions in L19 at position 40 (Q40H, Q40L and Q40R) caused a decrease in fitness while the G104A change had no effect on bacterial growth. The rate of protein synthesis was unaffected or increased by the mutations at position 40 and the level of read- through of a UGA nonsense codon was increased in vivo, indicating a loss of translational accuracy. The mutations in L19 increased sensitivity to aminoglycosides active at the A-site, further indicating a perturbation of the decoding step. These phenotypes are similar to those of the classical S4 and S5 ram (ribosomal ambiguity) mutants. By evolving low-fitness L19 mutants by serial passage, we showed that the fitness cost conferred by the L19 mutations could be compensated by additional mutations in the ribosomal protein L19 itself, in S12 and in L14, a protein located close to L19. Our results reveal a novel functional role for the 50 S ribosomal protein L19 during protein synthesis, supporting published structural data suggesting that the interaction of L14 and L19 with 16 S rRNA could influence function of the 30 S subunit. Moreover, our study demonstrates how compensatory fitness-evolution can be used to discover new molecular functions of ribosomal proteins. © 2006 Elsevier Ltd. All rights reserved. *Corresponding author Keywords: translation accuracy; translation rate; fitness; streptomycin resistance; compensatory evolution Introduction During the last four decades genetic, biochemical and structural analyses have shown that accurate decoding of mRNA involves several rRNA residues and ribosomal proteins. Antibiotics binding to the ribosome and analysis of bacterial mutants resistant to these antibiotics have been particularly useful in identifying the ribosomal decoding site and for understanding how different ribosomal components modulate fidelity. It was found earlier that presence of the aminoglycoside streptomycin allowed for the read-through of nonsense codons due to a general increase in translation errors. 1 In contrast, ribosomal mutants that reduce binding of aminoglycosides were found to increase the accuracy of tRNA selection (the hyper-accurate phenotype). For exam- ple, mutants resistant to the antibiotics neamine, gentamicin or streptomycin, that, respectively, have alterations in proteins S17 (rpsQ), L6 (rplF) and S12 (rpsL), show an increased proofreading efficiency for tRNA selection concomitantly with a decreased Abbreviation used: MIC, minimum inhibitory concentration. E-mail address of the corresponding author: smaisnierpatin@gmail.com doi:10.1016/j.jmb.2006.11.047 J. Mol. Biol. (2007) 366, 207–215 0022-2836/$ - see front matter © 2006 Elsevier Ltd. All rights reserved.