Genetic and structural analysis of base substitutions in the central pseudoknot of Thermus thermophilus 16S ribosomal RNA.

RNA
Steven T Gregory, Albert E Dahlberg

Abstract

Characterization of base substitutions in rRNAs has provided important insights into the mechanism of protein synthesis. Knowledge of the structural effects of such alterations is limited, and could be greatly expanded with the development of a genetic system based on an organism amenable to both genetics and structural biology. Here, we describe the genetic analysis of base substitutions in 16S ribosomal RNA of the extreme thermophile Thermus thermophilus, and an analysis of the conformational effects of these substitutions by structure probing with base-specific modifying agents. Gene replacement methods were used to construct a derivative of strain HB8 carrying a single 16S rRNA gene, allowing the isolation of spontaneous streptomycin-resistant mutants and subsequent genetic mapping of mutations by recombination. The residues altered to give streptomycin resistance reside within the central pseudoknot structure of 16S rRNA comprised of helices 1 and 27, and participate in the U13-U20-A915 base triple, the G21-A914 type II sheared G-A base pair, or the G885-C912 Watson-Crick base pair closing helix 27. Substitutions at any of the three residues engaged in the base triple were found to confer resistance. Results from structure...Continue Reading

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Citations

Oct 10, 2013·Biochimica Et Biophysica Acta·Ana MiguelJosé E Pérez-Ortín
May 1, 2013·Proceedings of the National Academy of Sciences of the United States of America·Crystal E FaganChristine M Dunham
Sep 18, 2013·Antimicrobial Agents and Chemotherapy·Cédric OrelleAlexander S Mankin
Feb 8, 2014·PloS One·Anton S PetrovLoren Dean Williams
Oct 19, 2016·PloS One·Jiajie HuangMichael Gribskov
Jan 22, 2015·Journal of Bacteriology·Jennifer F CarrSteven T Gregory

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