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

Abstract

Accurate tRNA selection by the ribosome is essential for the synthesis of functional proteins. Previous structural studies indicated that the ribosome distinguishes between cognate and near-cognate tRNAs by monitoring the geometry of the codon-anticodon helix in the decoding center using the universally conserved 16S rRNA bases G530, A1492 and A1493. These bases form hydrogen bonds with the 2’-hydroxyl groups of the codon-anticodon helix, which are expected to be disrupted with a near-cognate codon-anticodon helix. However, a recent structural study showed that G530, A1492 and A1493 form hydrogen bonds in an identical manner with both cognate and near-cognate codon-anticodon helices. To understand how the ribosome discriminates between cognate and near-cognate tRNAs, we made 2’-deoxynucleotide and 2’-fluoro substituted mRNAs, which disrupt the hydrogen bonds between the A site codon and G530, A1492 and A1493. Our results show that multiple 2’-deoxynucleotide substitutions in the mRNA substantially inhibit tRNA selection, whereas multiple 2’-fluoro substitutions in the mRNA have only modest effects on tRNA selection. Furthermore, the miscoding antibiotics paromomycin and streptomycin rescue the defects in tRNA selection with the multiple 2’-deoxynucleotide substituted mRNA. These results suggest that steric complementarity in the decoding center is more important than the hydrogen bonds between the A site codon and G530, A1492 and A1493 for tRNA selection.

Nucleotide modifications within bacterial messenger RNAs regulate their translation and are able to rewire the genetic code

“…These results are in line with earlier studies, reporting a 50–75%-fold decrease at distinct steps of translation (e.g. A-site tRNA binding or EF-Tu GTPase activation, respectively) when introducing single deoxy-nucleotides; an almost complete translational inhibition at the modified codon was observed introducing two or more deoxy-nucleotides ( 45 , 46 ).…”

New Structural Insights into Translational Miscoding

“…This longstanding concept has been difficult to demonstrate directly until recently, when the first structural evidence for errors in replication via GÁT and CÁA mismatches with Watson-Crick geometry was procured [60][61][62]. Other non-structural experimental findings also demonstrate that steric or shape complementarity, rather than H-bonding, is paramount in replication and translation [63][64][65][66].…”

Decoding on the ribosome depends on the structure of the mRNA phosphodiester backbone

“…What has been less understood is the extent to which the structure of the mRNA itself is important for this process. Nonetheless, biochemical studies of the role of the mRNA ribose backbone during decoding showed that the substitutions of A-site 2′-OH residues with deoxy or fluoro groups had minimal impact on tRNA selection (37). This is in contrast to structural studies, which showed that the hydroxyl groups are important for A-minor interactions with the decoding center nucleotides (31,32).…”

“…However, recent structural studies from a different group suggested that the hydrogen bonding was identical for both cognate and near-cognate codons (38) and that accuracy originates from energetic penalties associated with base-pair mismatches being forced to adopt a Watson-Crick base-pair geometry. Previous biochemical studies by Simpson and colleagues (37) explicitly addressed the role of the 2′-OH groups of the A site in decoding. Interestingly, single substitution of any of the 2′-OH groups by a deoxy only marginally affected peptide-bond formation.…”