UAG readthrough during TMV RNA translation: isolation and sequence of two tRNAsTyr with suppressor activity from tobacco plants

Abstract

The hypothetical replicase or replicase subunit cistron in the 5′-proximal part of tobacco mosaic virus (TMV) RNA yields a major 126-K protein and a minor 183-K ‘readthrough’ protein in vivo and in vitro. Two natural suppressor tRNAs were purified from uninfected tobacco plants on the basis of their ability to promote readthrough over the corresponding UAG termination codon in vitro. In a reticulocyte lysate the yield of 183-K readthrough protein increases from ˜10% in the absence of added tobacco plant tRNA up to ˜35% in the case of pure tRNA^Tyr added. Their amino acid acceptance and anticodon sequence (GψA) identifies the two natural suppressor tRNAs as the two normal major cytoplasmic tyrosine-specific tRNAs. tRNA^Tyr₁ has an A:U pair at the base of the TψC stem and an unmodified G₁₀, whereas tRNA^Tyr₂ contains a G:C pair in the corresponding location and m²G in position 10. This is the first case that, in a higher eukaryote, the complete structure is known of both the natural suppressor tRNAs and the corresponding viral RNA on which they exert their function. The corresponding codon-anticodon interaction, which is not in accordance with the wobble hypothesis, and the possible biological significance of the readthrough phenomenon is discussed.