Steric complementarity in the decoding center is important for tRNA selection by the ribosome

J Mol Biol. 2013 Oct 23;425(20):3778-89. doi: 10.1016/j.jmb.2013.02.038. Epub 2013 Mar 27.

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 ribosomal RNA 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 a manner identical with that of 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.

Keywords: EF-Tu; GTP hydrolysis; elongation factor Tu; kinetics; peptide bond; protein synthesis; rRNA; ribosomal RNA.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Anticodon
  • Codon
  • Escherichia coli / genetics
  • Escherichia coli / metabolism
  • Guanosine Triphosphate / chemistry
  • Guanosine Triphosphate / metabolism
  • Hydrolysis / drug effects
  • Nucleic Acid Conformation
  • Paromomycin / pharmacology
  • Peptide Elongation Factor Tu / chemistry
  • Peptide Elongation Factor Tu / metabolism
  • Protein Binding
  • Protein Biosynthesis / drug effects
  • Protein Biosynthesis / physiology
  • RNA, Messenger / chemistry
  • RNA, Messenger / genetics
  • RNA, Messenger / metabolism
  • RNA, Transfer / physiology*
  • Ribosomes / physiology*
  • Streptomycin / pharmacology

Substances

  • Anticodon
  • Codon
  • RNA, Messenger
  • Paromomycin
  • Guanosine Triphosphate
  • RNA, Transfer
  • Peptide Elongation Factor Tu
  • Streptomycin