The Brome mosaic virus subgenomic promoter hairpin is structurally similar to the iron-responsive element and functionally equivalent to the minus-strand core promoter stem-loop C

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Abstract
In the Bromoviridae family of plant viruses, trinucleotide hairpin loops play an important role in RNA transcription. Recently, we reported that Brome mosaic virus (BMV) subgenomic (sg) transcription depended on the formation of an unusual triloop hairpin. By native gel electrophoresis, enzymatic structure probing, and NMR spectroscopy it is shown here that in the absence of viral replicase the hexanucleotide loop 5′C1AUAG5A3′ of this RNA structure can adopt a pseudo trinucleotide loop conformation by transloop base pairing between C1 and G5. By means of in vitro replication assays using partially purified BMV RNA-dependent RNA polymerase (RdRp) it was found that other base pairs contribute to sg transcription, probably by stabilizing the formation of this pseudo triloop, which is proposed to be the primary element recognized by the viral replicase. The BMV pseudo triloop structure strongly resembles iron-responsive elements (IREs) in cellular messenger RNAs and may represent a general protein-binding motif. In addition, in vitro replication assays showed that the BMV sg hairpin is functionally equivalent to the minus-strand core promoter hairpin stem-loop C at the 3′ end of BMV RNAs. Replacement of the sg hairpin by stem-loop C yielded increased sg promoter activity whereas replacement of stem-loop C by the sg hairpin resulted in reduced minus-strand promoter activity. We conclude that AUA triloops represent the common motif in the BMV sg and minus-strand promoters required for recruitment of the viral replicase. Additional sequence elements of the minus-strand promoter are proposed to direct the RdRp to the initiation site at the 3′ end of the genomic RNA.