Quantitative Analysis of Deoxynucleotide Substitutions in the Codon–Anticodon Helix

Two Distinct Conformations of the Conserved RNA-rich Decoding Center of the Small Ribosomal Subunit Are Recognized by tRNAs and Release Factors

“…These data provide strong evidence for an essential role for these nucleotides in the process of tRNA selection. These data corroborate conclusions drawn from biochemical studies that probed the importance of interactions in this region by disrupting contacts within the codon:anticodon helix itself (Fahlman et al 2006;Gromadski et al 2006).…”

“…These data are consistent with the idea that mutation of the decoding center nucleotides destabilizes interactions with the codon:anticodon helix and that these defects can be suppressed by artificially inducing closure of the subunit with these compounds. Other related studies have similarly shown that these antibiotics can suppress a variety of defects in the decoding center including mismatches in the codon:anticodon helix and the loss of critical 2′-OH groups in this same region (Pape et al 2000;Fahlman et al 2006).…”

Steric Complementarity in the Decoding Center Is Important for tRNA Selection by the Ribosome

“…Previous studies have shown that 2’-deoxynucleotide substitutions in the A site codon inhibit the binding of aminoacylated and deacylated tRNA to the ribosomal A site 16; 17;18; 19 . However, these binding studies only examined the affinity of the tRNA in the fully accommodated A/A state on the ribosome.…”

“…Previous studies showed that single 2’-deoxynucleotide substitutions in A site codon inhibited the binding of tRNA to the A site by 3- to 4-fold 17; 18 , while 2’-deoxynucleotide substitution at all three positions in the A site codon reduced the tRNA binding affinity by 10-fold 16 . Since the A site tRNA does not directly contact the 2’-hydroxyl groups in the codon, these results are consistent with the idea that the loss of the 2’-hydroxyl groups from the A site codon inhibit “domain closure” and weakens the interaction of the tRNA with the ribosomal A site 17 . However, no studies have been carried out to systematically examine the role of these 2’-hydroxyl groups in the A site codon in individual steps of the tRNA selection pathway.…”

Stop Codon Recognition by Release Factors Induces Structural Rearrangement of the Ribosomal Decoding Center that Is Productive for Peptide Release

“…The decoding site nucleotides make four hydrogen-bonding interactions with 2 0 OH groups on the A site codon during tRNA selection (Ogle et al, 2001). On ribosomes carrying DNA in the A site, the loss of these stabilizing interactions between decoding site nucleotides and 2 0 OHs of the A site codon leads to a 10-fold reduction in the affinity of aminoacyl-tRNA (Fahlman et al, 2006;Potapov et al, 1995) and is thought to be responsible for failure to translate a DNA-based message (McCarthy and Holland, 1965). However, the magnitude of the defects in individual steps of tRNA selection had not been measured.…”