TRANSLATIONAL INITIATION AT THE COAT-PROTEIN GENE OF PHAGE MS2 - NATIVE UPSTREAM RNA RELIEVES INHIBITION BY LOCAL SECONDARY STRUCTURE

Maximal translation of the coat-protein gene from RNA bacteriophage MS 2 requires a contiguous stretch of native MS2 RNA that extends hundred s of nucleotides upstream from the translational start site. Deletion of these upstream sequences from MS2 cDNA plasmids results in a 30-fol d reduction of translational efficiency. By site-directed mutagenesis, we show that this low level of expression is caused by a hairpin stru cture centred around the initiation codon. When this hairpin is destab ilized by the introduction of mismatches, expression from the truncate d messenger increases 20-fold to almost the level of the full-length c onstruct. Thus, the translational effect of hundreds of upstream nucle otides can be mimicked by a single substitution that destabilizes the structure. The same hairpin is also present in full-length MS2 RNA, bu t there it does not impair ribosome binding. Apparently, the upstream RNA somehow reduces the inhibitory effect of the structure on translat ional initiation. The upstream MS2 sequence does not stimulate transla tion when cloned in front of another gene, nor can unrelated RNA segme nts activate the coat-protein gene. Several possible mechanisms for th e activation are discussed and a function in gene regulation of the ph age is suggested.