Synthetic RNA stem loops corresponding to positions 28–42
in the anticodon region of tRNAPhe bind efficiently
in an mRNA-dependent manner to ribosomes, whereas those
made from DNA do not. In order to identify the positions
where ribose is required, the anticodon stem-loop region
of tRNAPhe (Escherichia coli) was synthesized
chemically using a mixture of 2′-hydroxyl- and 2′-deoxynucleotide
phosphoramidites. Oligonucleotides whose ribose composition
allowed binding were retained selectively on nitrocellulose
filters via binding to 30S ribosomal subunits. The binding-competent
oligonucleotides were submitted to partial alkaline hydrolysis
to identify the positions that were enriched for ribose.
Quantification revealed a strong preference for a 2′-hydroxyl
group at position U33. This was shown directly by the 50-fold
lower binding affinity of a stem loop containing a single
deoxyribose at position U33. Similarly, defective binding
of the corresponding U33-2′-O-methyl-substituted
stem-loop RNA suggests that absence of the 2′-hydroxyl
group, rather than an altered sugar pucker, is responsible.
Stem-loop oligoribonucleotides from different tRNAs with
U33-deoxy substitutions showed similar, although quantitatively
different effects, suggesting that intramolecular rather
than tRNA-ribosome interactions are affected. Because the
2′-hydroxyl group of U33 was shown to be a major
determinant of the U-turn of the anticodon loop in the
crystal structure of tRNAPhe in yeast, our finding
might indicate that the U-turn conformation in the anticodon
loop is required and/or maintained when the tRNA is
bound to the ribosomal P site.