Anticodon Domain Modifications Contribute Order to tRNA for Ribosome- Mediated Codon Binding super([dagger]) super([dagger])

• Published in: Biochemistry (Easton) Vol. 47; no. 23; pp. 6117 - 6129
• Main Authors: Graham, William D, Agris, Paul F, Sproat, Brian, Gustilo, Estella M, Malkiewicz, Andrzej, Dziergowska, Agnieszka, Vendeix, Franck AP
• Format: Journal Article
• Language: English
• Published: 01.01.2008
• ISSN: 0006-2960
• DOI: 10.1021/bi702356j

The accuracy and efficiency with which tRNA decodes genomic information into proteins require posttranscriptional modifications in or adjacent to the anticodon. The modification uridine-5-oxyacetic acid (cmo super(5)U sub(34)) is found at wobble position 34 in a single isoaccepting tRNA species for six amino acids, alanine, leucine, proline, serine, threonine, and valine, each having 4-fold degenerate codons. cmo super(5)U sub(34) makes possible the decoding of 24 codons by just six tRNAs. The contributions of this important modification to the structures and codon binding affinities of the unmodified and fully modified anticodon stem and loop domains of tRNA super(Val3) sub(UAC) (ASL super(Val3) sub(UAC)) were elucidated. The stems of the unmodified ASL super(Val3) sub(UAC) and that with cmo super(5)U sub(34) and N super(6)- methyladenosine, m super(6)A sub(37), adopted an A-form RNA conformation (rmsd similar to 0.6 Aa) as determined with NMR spectroscopy and torsion-angle molecular dynamics. However, the UV hyperchromicity, circular dichroism ellipticity, and structural analyses indicated that the anticodon modifications enhanced order in the loop. ASL super(Val3) sub(UAC)-cmo super(5)U sub(34); m super(6)A sub(37) exhibited high affinities for its cognate and wobble codons GUA and GUG, and for GUU in the A-site of the programmed 30S ribosomal subunit, whereas the unmodified ASL super(Val3) sub(UAC) bound less strongly to GUA and not at all to GUG and GUU. Together with recent crystal structures of ASL super(Val3) sub(UAC)- cmo super(5)U sub(34); m super(6)A sub(37) bound to all four of the valine codons in the A-site of the ribosome's 30S subunit, these results clearly demonstrate that the xo super(5)U sub(34)-type modifications order the anticodon loop prior to A- site codon binding for an expanded codon reading, possibly reducing an entropic energy barrier to codon binding.