Studies on the mechanism of translational enhancement by the 5′‐leader sequence of tobacco mosaic virus RNA

Translation of foreign mRNAs is enhanced by a cis‐acting derivative (Ω′) of the 5′‐leader sequence (Ω) of tobacco mosaic virus RNA (vulgare strain). To explain this effect we have conducted several experiments in vitro. The presence of various 5′‐terminal sequences, including Ω′, did not significantly increase the half‐lives of chloramphenicol acetyltransferase (CAT) or neomycin phosphotransferase (NPTII) mRNAs in wheat‐germ extract. Also, a long leader sequence, unrelated to Ω', did not enhance expression of NPTII mRNA in vitro. The ability of several leader sequences, including Ω′, to form multiple initiation complexes with 80S (wheat germ) ribosomes was examined using CAT or NPTII mRNAs incubated in the presence of sparsomycin. Formation of disome complexes was unrelated to the capacity of a 5′‐leader sequence to enhance translation. Expression of CAT mRNA in both wheat germ extract and messenger‐dependent rabbit reticulocyte lysate was less susceptible to inhibition by increasing salt concentration when a 5′‐proximal Ω′ sequence was present. This effect was less marked when the CAT mRNA was capped. Conversely at high salt concentrations, capping was less stimulatory for mRNA with a 5′‐proximal Ω′ sequence. These data suggest that Ω′ and the cap enhance translation, at least in part, by a similar mechanism. We propose that both features reduce RNA secondary structure, thereby rendering the 5′ terminus more accessible to scanning by 40s ribosomal subunits and/or interaction with associated initiation factors. This conclusion was supported by computer‐based secondary‐structure analyses of our SP6 RNA polymerase transcript sequences. The ability of 5′ leader sequences from brome mosaic virus RNA 3, alfalfa mosaic virus RNA 4, and the genomic RNAs of turnip yellow mosaic virus, Rous sarcoma virus or tobacco mosaic virus (tomato strain) to enhance mRNA translation in eukaryotic systems may also be correlated with their respective secondary structures. A different mechanism probably accounts for the Ω′‐dependent enhancement of mRNA expression in Escherichia coli or in E. coli cell‐free systems.