Mutagenesis at the mRNA decoding site in the 16S ribosomal RNA using the specialized ribosome system in Escherichia coli

Abstract

In the specialized ribosome system, a distinct pool of mutated ribosomes is dedicated to the translation of one particular mRNA species. This was accomplished by altering the Shine-Dalgarno sequence on the mRNA and its complementary anti-Shine-Dalgarno sequence on the plasmid-borne 16S rRNA gene. Here, using the specialized ribosome system, we were able to introduce mutations in key regions of the 16S rRNA and could study their effect on translation in vivo. The C1400 region has been implicated to play a role in the actual mRNA decoding process. Several ribosomal mutations were introduced in this region. We showed that substitution of the evolutionary highly conserved C1400 residue by a G- or an A-residue inhibits ribosomal activity by 80% and 50% respectively, whereas, a C to a U change at this conserved position does not affect overall ribosomal activity. The adjacent stem structure (1410-1490) was also examined. Disruption of the stem by replacing either one of the arms of this stem, with a different sequence, inhibits ribosomal activity by approximately 80%. A small but significant restoration of translation could be achieved by recreating a complementary stem with a different sequence. We found that full reversion of activity could be obtained when such mutated ribosomes were made spectinomycin resistant by introducing a C to A substitution at position 1192 which is located far away in the secondary structure map of the 16S rRNA molecule. Based on these results we conclude that some, but not all, of the nucleotides in the conserved C1400 region play a key role in translation.(ABSTRACT TRUNCATED AT 250 WORDS)