IDENTIFICATION OF A COMPETITIVE TRANSLATION DETERMINANT IN THE 3'-UNTRANSLATED REGION OF ALFALFA MOSAIC-VIRUS COAT PROTEIN MESSENGER-RNA

We report that the competitive translational activity of alfalfa mosai c virus coat protein mRNA (CP RNA), a nonadenylated mRNA, is determine d in part by the 3' untranslated region (UTR). Competitive translation was characterized both in vitro, with cotranslation assays, and in vi vo, with microinjected Xenopus laevis oocytes. In wheat germ extracts, coat protein synthesis was constant when a fixed amount of full-lengt h CP RNA was cotranslated with increasing concentrations of competitor globin mRNA. However, translation of CP RNA lacking the 3' UTR decrea sed significantly under competitive conditions. RNA stabilities were e quivalent. In X. laevis oocytes, which are translationally saturated a nd are an inherently competitive translational environment, full-lengt h CP RNA assembled into large polysomes and coat protein synthesis was readily detectable. Alternatively, CP RNA lacking the 3' UTR sediment ed as small polysomes, and little coat protein was detected. Again, RN A stabilities were equivalent. Site-directed mutagenesis was used to l ocalize RNA sequences or structures required for competitive translati on. Since the CP RNA 3' UTR has an unusually large number of AUG nucle otide triplets, two AUG-containing sites were altered in full-length R NA prior to oocyte injections. Nucleotide substitutions at the sequenc e GAUG, 20 nucleotides downstream of the coat protein termination codo n, specifically reduced full-length CP RNA translation, while similar substitutions at the next AUG triplet had little effect on translation . The competitive influence of the 3' UTR could be explained by RNA-pr otein interactions that affect translation initiation or by ribosome r einitiation at downstream AUG codons, which would increase the number of ribosomes committed to coat protein synthesis.