Expression of a plant viral polycistronic mRNA in yeast, Saccharomyces cerevisiae, mediated by a plant virus translational transactivator.

Abstract

We demonstrate that the cauliflower mosaic virus (CaMV) gene VI product can transactivate the expression of a reporter gene in bakers' yeast, Saccharomyces cerevisiae. The gene Vlcoding sequence was placed under the control of the galactose-inducible promoter GALI, which is present in the yeast shuttle vector pYES2, to create plasmid JS169. We also created a chloramphenicol acetyltransferase (CAT) reporter plasmid, JS161, by inserting the CAT reporter gene in-frame into CaMV gene II and subsequently cloning the entire CaMV genome into the yeast vector pRS314. When JS161 was transformed into yeast and subsequently assayed for CAT activity, only a very low level of CAT activity was detected in cellular extracts. To investigate whether the CaMV gene VI product would mediate an increase in CAT activity, we cotransformed yeast with JS169 and JS161. Upon induction with galactose, we found that CAT activity in yeast transformed with JS161 and JS169 was about 19 times higher than the level in the transformants that contained only JS161. CAT activity was dependent on the presence of the gene VI protein, because essentially no CAT activity was detected in yeast cells grown in the presence of glucose, which represses expression from the GAL) promoter. RNase protection assays showed that the gene VI product had no effect on transcription from the 35S RNA promoter, demonstrating that regulation was occurring at the translation level. This yeast system will prove useful for understanding how the gene VI product of CaMV mediates the translation of genes present on a eukaryotic polycistronic mRNA.Cauliflower mosaic virus (CaMV) is a plant pararetrovirus that has a unique strategy for the expression of its six genes. Two RNAs that have sizes of 19S and 35S are transcribed from the circular double-stranded DNA template (1). The monocistronic 19S RNA is responsible for the expression of the CaMV gene VI product, a protein that has been described as a translational transactivator (2). The 35S RNA is a terminally redundant RNA that has dual roles in replication and expression. It serves as a replication intermediate during reverse transcription of the viral genome (1). In addition, it is a polycistronic mRNA that contains a 600-nucleotide leader sequence followed by six closely spaced genes, including gene VI (1). The 35S RNA leader sequence itself is extremely complex. It contains seven to eight small open reading frames, depending on the strain, that vary in size from 9 to 102 nucleotides; it may be folded into an extensive stem-loop structure (3); and it inhibits translation of genes I-V in transiently transfected protoplasts (4, 5).The unusual feature about the CaMV expression strategy is that the CaMV gene VI protein is necessary for efficient translation of genes I-Von the 35S RNA (6-9). Genes I-V, and possibly VI, are believed to be expressed by translation reinitiation; as ribosomes complete the translation of one gene, they reinitiate protein synthesis at the next start codon until all genes are translated (10-12). ...

Advancing secondary metabolite biosynthesis in yeast with synthetic biology tools

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