Single atom modification (O → S) of tRNA confers ribosome binding

S. Salman Ashraf, Elzbieta Sochacka, Robert Cain, Richard Guenther, Andrzej Malkiewicz, Paul F. Agris

Research output: Contribution to journalArticlepeer-review

123 Citations (Scopus)

Abstract

Escherichia coli tRNA(Lys)(SUU), as well as human tRNA(Lys3)(SUU), has 2-thiouridine derivatives at wobble position 34 (s2U*34). Unlike the native tRNA(Lys)(SUU), the full-length, unmodified transcript of human tRNA(Lys3)(UUU) and the unmodified tRNA(Lys3)(UUU) anticodon stem/loop (ASL(Lys3)(UUU)) did not bind AAA- or AAG-programmed ribosomes. In contrast, the completely unmodified yeast tRNA(Phe) anticodon stem/loop (ASL(Phe)(GAA)) had an affinity (K(d) = 136 ± 49 nM) similar to that of native yeast tRNA(Phe)(GmAA) (K(d) = 103 ± 19 nM). We have found that the single, site- specific substitution of s2U34 for U34 to produce the modified ASL(Lys)(SUU) was sufficient to restore ribosomal binding. The modified ASL(Lys)(SUU) bound the ribosome with an affinity (K(d) = 176 ± 62 nM) comparable to that of native tRNA(Lys)(SUU) (K(d) = 70 ± 7 nM). Furthermore, in binding to the ribosome, the modified ASL(Lys3)(SUU) produced the same 16S P-site tRNA footprint as did native E. coli tRNA(Lys)(SUU), yeast tRNA(Phe)(GmAA), and the unmodified ASL(Phe)(GAA). The unmodified ASL(Lys3)(UUU) had no footprint at all. Investigations of thermal stability and structure monitored by UV spectroscopy and NMR showed that the dynamic conformation of the loop of modified ASL(Lys3)(SUU) was different from that of the unmodified ASL(Lys)(UUU), whereas the stems were isomorphous. Based on these and other data, we conclude that s2U34 in tRNA(Lys)(SUU) and in other s2U34-containing tRNAs is critical for generating an anticodon conformation that leads to effective codon interaction in all organisms. This is the first example of a single atom substitution (U34 → s2U34) that confers the property of ribosomal binding on an otherwise inactive tRNA.

Original languageEnglish
Pages (from-to)188-194
Number of pages7
JournalRNA
Volume5
Issue number2
DOIs
Publication statusPublished - Feb 1999
Externally publishedYes

Keywords

  • 2-thiouridine
  • Anticodon conformation
  • Anticodon stem/loop analogs
  • Nucleoside modifications
  • Ribosome
  • Translation
  • tRNA(Lys)

ASJC Scopus subject areas

  • Molecular Biology

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