Identification of specific Rp-phosphate oxygens in the tRNA anticodon loop required for ribosomal P-site binding

Abstract

ABSTRACTtRNA binding to the ribosomal P site is dependent not only on correct codon-anticodon interaction but also involves identification of structural elements of tRNA by the ribosome. By using a phosphorothioate substitutioninterference approach, we identified specific nonbridging Rp-phosphate oxygens in the anticodon loop of tRNA Phe from Escherichia coli which are required for P-site binding. Stereospecific involvement of phosphate oxygens at these positions was confirmed by using synthetic anticodon arm analogues at which single Rp-or Sp-phosphorothioates were incorporated. Identical interference results with yeast tRNA Phe and E. coli tRNA f Met indicate a common backbone conformation or common recognition elements in the anticodon loop of tRNAs. N-ethyl-N-nitrosourea modification-interference experiments with natural tRNAs point to the importance of the same phosphates in the loop. Guided by the crystal structure of tRNA Phe , we propose that specific Rp-phosphate oxygens are required for anticodon loop (''U-turn'') stabilization or are involved in interactions with the ribosome on correct tRNAmRNA complex formation.

Non-bridging Phosphate Oxygen Atoms within the tRNA Anticodon Stem-loop are Essential for Ribosomal A Site Binding and Translocation

“…This correlates well with work that employed various techniques to show that the backbone at position 33 is protected by the ribosome at the P site. 14,15 However, due to the diminished level of translocation observed at the lower concentration, it is difficult to determine if the inhibition observed in the translocation assay is a result of the disruption of an interaction with 16 S rRNA base U1341 during translocation to the P site, or the disruption of an interaction with 16 S rRNA base G963 during binding to the A site.…”

“…12,13 The incorporation of phosphorothioate has little effect on overall tRNA structure or ribosomal binding, unless the phosphorothioate interferes specifically with a functionally relevant interaction. 14,15 Here, we present the first systematic examination of the importance of non-bridging phosphate oxygen atoms within the anticodon stem-loop of tRNA for binding to the A site of the ribosome and translocation to the P site. We utilize both transcript and synthetic methods for the complete incorporation of phosphorothioate at specific positions in the anticodon stem-loop and assess their effect on A site binding and translocation using a previously established in vitro translocation assay.…”

Nonbridging phosphate oxygens in 16S rRNA important for 30S subunit assembly and association with the 50S ribosomal subunit

“…The phosphorothioate-substituted 16S rRNA from the total and selected population was cleaved using iodine to identify the location of individual phosphorothioate substitutions within the 16S rRNA (Schnitzer and von Ahsen 1997). About 40 µg of purified 16S rRNA was incubated in 80% formamide and 2 mM iodine (final concentration) in 100 µL of total reaction volume for 2 min at room temperature.…”

Recognition Elements in rRNA for the Tylosin Resistance Methyltransferase RlmAII

“…Substitution of the G748 pro-R p phosphate oxygen atom increases the binding affinity of RlmA II , an observation also made for position 34 in tRNA Phe where introduction of a phosphorothiate increases binding affinity for the ribosomal P site. 33 This could be one of the defining differences between the RNA interaction of RlmA II and that of its Gram-negative ortholog, RlmA I . Both methyltransferases recognize the same three-helix junction in the rRNA with subtle yet important distinctions, which guide them to their respective targets at G745 for RlmA I and at G748 for RlmA II .…”