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Helix 69 in 23S rRNA modulates decoding by wild type and suppressor tRNAs

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Abstract

Helix 69 of 23S rRNA forms one of the major inter-subunit bridges of the 70S ribosome and interacts with A- and P-site tRNAs and translation factors. Despite the proximity of h69 to the decoding center and tRNAs, the contribution of h69 to the tRNA selection process is unclear: previous genetic analyses have shown that h69 mutations increase frameshifting and readthrough of stop codons. However, a complete deletion of h69 does not affect the selection of cognate tRNAs in vitro. To address these discrepancies, the in vivo effects of a range of single- and multi-base h69 mutations in Escherichia coli 23S rRNA on various translation errors have been determined. While a majority of the h69 mutations examined here affected readthrough of stop codons and frameshifting, the ΔA1916 single base deletion mutation uniquely influenced missense decoding. Different h69 mutants had either increased or decreased levels of stop codon readthrough. The h69 mutations that decreased UGA readthrough also decreased UGA reading by a mutant, near-cognate tRNATrp carrying a G24A substitution in the D arm, but had far less effect on UGA reading by a suppressor tRNA with a complementary anticodon. These results suggest that h69 interactions with release factors contribute significantly to termination efficiency and that interaction with the D arm of A-site tRNA is important for discrimination between cognate and near-cognate tRNAs.

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Acknowledgments

I am grateful to Dr. James Curran, Wake Forest University for providing the trpT mutant plasmids, to Drs. Catherine Squires and Selwyn Quan for supplying the MG1655-derived Δ7 prrn strain and to Dr. Qing Sun for constructing some of the lacZ plasmids. Thanks are due to Drs. Steven Gregory and Aivar Liiv for their comments on the manuscript. This work was supported by grants # MCB 0343942 and MCB0745025 from the National Science Foundation.

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  1. School of Biological Sciences, University of Missouri-Kansas City, 5007 Rockhill Rd., Kansas City, MO, 64110, USA

    Michael O’Connor

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  1. Michael O’Connor
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Correspondence to Michael O’Connor.

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Communicated by S. Hohmann.

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O’Connor, M. Helix 69 in 23S rRNA modulates decoding by wild type and suppressor tRNAs. Mol Genet Genomics 282, 371–380 (2009). https://doi.org/10.1007/s00438-009-0470-6

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