Mutations of Bacteriophage T7 That Affect Initiation of Synthesis of the Gene 0.3 Protein

Two different mutations that greatly diminish the rate of synthesis of the gene 0.3 protein of bacteriophage T7 have been characterized. One is in the initiator triplet for the 0.3 protein, changing it from AUG to ACG. This mutation was found to have little effect on binding of ribosomes to the 0.3 mRNA in vitro, although 0.3 protein synthesis was greatly depressed in vitro as well as in vivo. A suppressor mutation that partially restores the wild-type rate of synthesis was found to lie within the 0.3 RNA but not close to the mutant ACG (more than 64 nucleotides away). The second mutation is a G-to-A transition located 11 bases to the 5' side of the initiator AUG. This change eliminates a possible five-base pairing with a sequence near the 3' end of 16S ribosomal RNA, an interaction previous workers have proposed to be important for initiation of protein synthesis. This mutation causes the site of ribosome binding to shift about 15 bases to the 3' side, centering on an internal AUG, but this new site has only a poor potential interaction with 16S RNA. A suppressor mutation that restores the rate of 0.3 protein synthesis to essentially wild-type levels and also restores wild-type ribosome-binding behavior was found to lie adjacent to the original mutation, creating a new four-base complementarity with 16S RNA. These results provide strong support for the idea that a pairing interaction between mRNA and 16S RNA is involved in specific initiation of protein synthesis in Escherichia coli and indicate that this interaction may be important in selecting the site in mRNA at which the ribosomes bind.