A 5′-terminal phosphate is required for stable ternary complex formation and translation of leaderless mRNA in Escherichia coli

Mapping of mRNA 5′-ends by primer extension analysis of leadered (lanes 1,2) and leaderless (lanes 3,4) cI-lacZ fusions with a 5′-terminal hydroxyl or triphosphate. The closed arrows identify the 5′-end of the lac-untranslated leader (lanes 1,2) or the 5′-end (i.e., the translational start site) of LL mRNAs (lanes 3,4). The open arrows identify the expected migratory positions of uncleaved, hammerhead-containing mRNA. Lanes 2 and 3 show in vivo mRNA containing a 5′-hydroxyl, and lanes 1 and 4 show in vivo mRNA containing a 5′-triphosphate. Lanes labeled G, A, T, and C indicate the dideoxy termination sequencing reactions used to map the 5′ ends of transcripts.