The nucleotides guanine 530 (G530) and adenine 1492 (A1492) of 16S ribosomal RNA (rRNA) are essential for discrimination of tRNA in the A site. These bases undergo conformational changes to form hydrogen bonds with the minor groove of the codon-anticodon complex. These hydrogen bonds are crucial for selection of the correct tRNA species. G530 and A1492 not only hydrogen bond with the codon-anticodon complex but they also hydrogen bond with each other. Lethality associated with mutations in G30 and A1492 could be attributed to the importance of their interaction with each other. These two residues have never been mutated together but it has been suggested through computer simulations that a G530 to adenine and A1492 guanine (G530A-A1492G) double mutant may be viable. The mutations have been created and assessed for their ability to maintain viability and translate reporter genes.

The nucleotide following the stop codon UGA has been implicated to have a role in recognition of termination codons by prokaryotic release factor 2. Mutations in the nucleotide following UGA (fourth base) cause a reduction in stop codon recognition (suppression). Statistical analysis and experimental evidence shows that recognition dependent on the nucleotide following UGA has the hierarchy U>G>A>C. Mutations in the cytosine 1054 (C1054) of 16S ribosomal RNA also confer UGA suppression, but the hierarchy for stop codon recognition hasn’t been established. Structural mapping and crosslinking analysis have shown that the nucleotide following UGA and C1054 are in close proximity within the ribosome. However, their effects on UGA stop codon suppression have not been studied together. Through mutagenesis of both the 1054 position of 16S and the nucleotide following UGA, I am examining termination efficiency by altering the fourth nucleotide and the identity of position 1054.