title: 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 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.
authors.affiliation: Department of Chemistry and Biochemistry, University of California San Diego, 9500 Gilman Drive, La Jolla, CA 92093-0314, USA.
authors.forename: Prashant K
authors.initials: PK
authors.lastname: Khade
authors.forename: Xinying
authors.initials: X
authors.lastname: Shi
authors.forename: Simpson
authors.initials: S
authors.lastname: Joseph
mesh_terms: D000926:Anticodon; D003062:Codon; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000737:chemistry / Q000378:metabolism; D006868:Hydrolysis / Q000187:drug effects; D009690:Nucleic Acid Conformation; D010303:Paromomycin / Q000494:pharmacology; D010444:Peptide Elongation Factor Tu / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding; D014176:Protein Biosynthesis / Q000187:drug effects / Q000502:physiology; D012333:RNA, Messenger / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012343:RNA, Transfer / Q000502:physiology*; D012270:Ribosomes / Q000502:physiology*; D013307:Streptomycin / Q000494:pharmacology
chemical_list: D000926:Anticodon; D003062:Codon; D012333:RNA, Messenger; D010303:Paromomycin; D006160:Guanosine Triphosphate; D012343:RNA, Transfer; D010444:Peptide Elongation Factor Tu; D013307:Streptomycin
keywords: EF-Tu; GTP hydrolysis; elongation factor Tu; kinetics; peptide bond; protein synthesis; rRNA; ribosomal RNA
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