Structural studies of the ribosome have benefited greatly from the use of organisms adapted to extreme environments. However, little is known about the mechanisms by which ribosomes or other ribonucleoprotein complexes have adapted to functioning under extreme conditions, and it is unclear to what degree mutant phenotypes of extremophiles will resemble those of their counterparts adapted to more moderate environments. It is conceivable that phenotypes of mutations affecting thermophilic ribosomes, for instance, will be influenced by structural adaptations specific to a thermophilic existence. This consideration is particularly important when using crystal structures of thermophilic ribosomes to interpret genetic results from nonextremophilic species. To address this issue, we have conducted a survey of spontaneously arising antibioticresistant mutants of the extremely thermophilic bacterium Thermus thermophilus, a species which has featured prominently in ribosome structural studies. We have accumulated over 20 single-base substitutions in T. thermophilus 16S and 23S rRNA, in the decoding site and in the peptidyltransferase active site of the ribosome. These mutations produce phenotypes that are largely identical to those of corresponding mutants of mesophilic organisms encompassing a broad phylogenetic range, suggesting that T. thermophilus may be an ideal model system for the study of ribosome structure and function.Members of the bacterial genus Thermus are extreme thermophiles first described by Brock and Freeze in 1969 (2) and have since been found in terrestrial and marine thermal environments throughout the world (60). Together with Deinococcus, Meiothermus, Marinithermus, Oceanithermus, and Vulcanithermus, they form a deeply branching phylum now known to be monophyletic (23,59). The close affiliation between Thermus and Deinococcus has been confirmed by complete genome sequences of Thermus thermophilus (30) and Deinococcus radiodurans (59). The finding of slightly thermophilic species related to Deinococcus, together with the thermophilic nature of the other genera of this phylum, suggests that thermophily is a primitive character of this clade (59).Recent advances in structural biology have produced a vast reservoir of high-resolution structural information regarding components of the protein synthetic machinery from members of the Deinococcus-Thermus phylum, including high-resolution crystal structures of the T. thermophilus 30S subunit (47, 61), medium-resolution structures of the entire T. thermophilus 70S ribosome (65), and high-resolution structures of the D. radiodurans 50S subunit (26). The value of these structures is magnified by their interpretation in light of several decades of genetics and biochemistry using ribosomes from mesophiles such as Escherichia coli. However, the ability to make such interpretations is potentially compromised by the absence of genetic and biochemical data obtained for ribosomes from members of the Deinococcus-Thermus phylum. It is assumed that nucleotide sequence c...