Identification of 2'-hydroxyl groups required for interaction of a tRNA anticodon stem-loop region with the ribosome.
Synthetic RNA stem loops corresponding to positions 28-42 in the anticodon region of tRNA(Phe) 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 tRNA(Phe) (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 tRNA(Phe) 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.
von Ahsen
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Institute of Microbiology and Genetics, University of Vienna, Austria. ahsen@nt.imp.univie.ac.at
Green
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Schroeder
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Noller
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D000926:Anticodon / Q000138:chemical synthesis / Q000737:chemistry / Q000378:metabolism*; D001665:Binding Sites; D003062:Codon / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics; D009690:Nucleic Acid Conformation; D012329:RNA, Bacterial / Q000138:chemical synthesis / Q000737:chemistry / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism; D012360:RNA, Transfer, Phe / Q000138:chemical synthesis / Q000737:chemistry / Q000378:metabolism*; D012270:Ribosomes / Q000378:metabolism*; D013329:Structure-Activity Relationship
D000926:Anticodon; D003062:Codon; D012329:RNA, Bacterial; D012333:RNA, Messenger; D012360:RNA, Transfer, Phe