Summary
Bacteriophage T7 0.3 mRNA synthesised and processed in vitro has been purified starting from the DNA of T7+ as well as from that of two initiation mutants of T7 (CR17 with a U → C transition in the initiation codon and CR35b whose potential Shine and Dalgarno (S-D) interaction is interrupted by a G → A transition). These mRNAs were used as templates to direct the binding of fMet-tRNA and the synthesis of 0.3 protein in both E. coli and wheat germ cell-free systems. The initiation codon mutant displayed approximately 50% inhibition of fMet-tRNA binding and 0.3 protein synthesis in both systems. The S-D sequence mutant, on the other hand, was found to be less affected than the initiation triplet mutant (20%–40% inhibition) in both fMet-tRNA binding and template activity in the E. coli system. In the wheat germ system, which does not make use of the S-D interaction, however, this mutant displayed normal template activity suggesting that the inhibition obtained in the E. coli system, albeit slight, is due to the impairment of the S-D interaction and not to an alteration of the mRNA secondary or tertiary structure caused by the base substitution.
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Ohsawa, H., Herrlich, P. & Gualerzi, C. In vitro template activity of 0.3 mRNA from wild type and initiation mutants of bacteriophage T7. Mol Gen Genet 196, 53–58 (1984). https://doi.org/10.1007/BF00334091
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DOI: https://doi.org/10.1007/BF00334091