Analysis of Mutations in the 23S rRNA
Several experimental approaches have been employed to study the structure and function of ribosomes (reviewed in Dahlberg, 1989, and Leclerc and Brakier-Gingras, 1990). One such approach is that of site-directed mutagenesis of the rRNA, most commonly in the rRNA of the bacterium Escherichia coli. This organism has seven rRNA operons and thus separation of the host encoded rRNA from that of the mutated plasmid-encoded rRNA is necessary for further biochemical studies. The problem can be overcome by using in vitro synthesized rRNA for reconstitution of the subunit. This has been sucessfully achieved for total reconstitution of functional 30S particles. In contrast, transcribed and unmodified 23S rRNA cannot form active particles in reconstitution assays. Therefore, 23S rRNA should be expressed in vivo followed by isolation of the ribosomes. It is possible to discriminate between activities of mutant and wild-type ribsomes by using a second mutation in the gene of 23S rRNA. One such mutation is the A to T transversion corresponding to the position 1067 of 23S rRNA which confers resistance to thiostrepton during cell-free translation (Thompson et al., 1988). Thiostrepton has a high affinity to wild-type ribosomes. U1067 ribosomes bind the drug with a reduced affinity (Thompson and Cundliffe, 1991). Therefore, A to U substitution at position 1067 can be used for selective inactivation of wild-type ribosomes and for physical separation of mutant ribosomes. This experimental approach has been used to analyze functional properties of mutations corresponding to positions G2505 and G2583 of 23S rRNA (Saarma and Remme, 1992). The most interesting finding was that mutations at position 2583 increased the translational accuracy. These results, together with those of mutations at positions G2505, G2583, G2607, and G2608 in vitro and G2505 and G2583 in vivo will be summarized here.
KeywordsPeptide Bond Formation Translational Accuracy Mutant Ribosome Thiostrepton Resistance Transcription Initiation Rate
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