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

Competition primer extension inhibition (toeprint) analysis of LL cI-lacZ mRNA bound to 70S ribosomes with or without tRNA^fMet. Prebound ternary complexes containing LL cI-lacZ mRNA with a 5′-monophosphate (A) or 5′-hydroxyl (B) are challenged with excess competitor mRNA containing a 5′-hydroxyl or a 5′-monophosphate. (A) Stability of ternary complexes containing 5′-phosphate LL mRNA. Lane 1 excludes ribosomes, and lane 2 excludes tRNA^fMet. (Lanes 3–5) The ternary complex incubated for 0, 20, and 30 min after the initial binding, respectively, with no addition of competitor mRNA. (Lanes 6–10) A 5′-hydroxyl LL mRNA competitor (20X) was added 15 min after initial ternary complex formation with primer-annealed 5′phosphate mRNA (1X). (Lanes 11–15) A 5′-phosphate LL competitor mRNA (20X) was added 15 min after initial ternary complex formation with primer-annealed 5′phosphate mRNA (1X). Reverse transcription was initiated at 0, 5, 10, 20, or 30 min after the competitor addition. (B) Stability of ternary complexes containing 5′-hydroxyl LL mRNA. Lane 1 excludes ribosomes, and lane 2 excludes tRNA^fMet. (Lanes 3,4) The ternary complex incubated for 0 and 20 min, respectively, after the complex formation, with no addition of competitor mRNA. (Lanes 5–8) A 5′-hydroxyl LL mRNA competitor (20X) was added to the reactions; (lanes 9–12) a 5′-phosphate LL mRNA competitor (20X) was added 15 min after initial ternary complex formation with primer-annealed 5′hydroxyl mRNA (1X). Reverse transcription was initiated at 0, 5, 10, and 20 min after the competitor addition. The position of the toeprint signals are identified by arrows.