The 5'-leader sequence of tobacco mosaic virus RNA enhances the expression of foreign gene transcripts<i>in vitro</i>and<i>in vivo</i>

TLDR

A 67‑nt 5′‑leader sequence of tobacco mosaic virus RNA (Ω′) enhances translation of foreign genes in both in vitro and in vivo systems. The authors inserted chemically synthesized Ω′ between the SP6 promoter and reporter genes in a plasmid, generated run‑off transcripts with or without a 5′‑cap and Ω′, and evaluated them in rabbit reticulocyte, wheat germ, and E. coli cell‑free translation systems, as well as by electroporating tobacco protoplasts and microinjecting Xenopus oocytes.

Abstract

A 67-nucleotide portion of the non-coding, 5′-leader sequence of tobacco mosaic virus RNA [defined as Ω' (Gr. omega prime)] has been shown to enhance the translation of contiguous foreign gene transcripts both in vitro and in vivo. Chemically-synthesized Ω', containing convenient linker sequences, was inserted into derivatives of an in vitro transcription plasmid (pSP64) between the bacteriophage-SP6 promoter and sequences coding for either chloramphenicol acetyltransferase (CAT) or neomycin phosphotransferase (NPTII). Run-off in vitro transcripts, with or without a 5′-cap structure (G(5′)ppp(5′)G) and/or the Ω' sequence, were tested in mRNA-dependent cell-free translation systems derived from rabbit reticulocyte lysate, wheat germ extract or Escherichia coli (MRE 600). In all cases, the presence of Ω' increased the translational expression of both reporter genes, typically between 2- to 10-fold. Electroporation of isolated mesophyll protoplasts from Nicotiana tabacum cv. Xanthi, or microinjection of oocytes from Xenopus laevis, with SP6-transcripts containing the CAT-coding region confirmed and extended the value of Ω' as a potential translational enhancer of gene expression in vivo.