The mRNA is bound and poised in the decoding center of the small subunit of the ribosome where the genetic code is translated by the tRNAs, which recognize sense codons, and by the release factors, which recognize stop-codons. Structural and biochemical studies have identified key universally conserved nucleotides, G530, A1492, and A1493, that are important for selection of cognate tRNA species during elongation. Here, we present evidence that these same universally conserved nucleotides are also important for interactions with the release factors, but must assume a very different structure during stopcodon recognition. These data provide mechanistic insight into how the decoding center of the ribosome has evolved to recognize distinct substrates with high fidelity, which in turn regulates the downstream chemical events of peptidyl transfer and peptide release.