Rather than have the SV40 PolyA sequence and 3' elements 5' to 3' on the coding strand, many reporeter expression vectors (such as EGFP) have the PolyA 5' to 3' on the opposite strand (with the end of the 3' element nearest the stop codon and the start of the PolyA furthest away). How does this influence polyadenylation of the reporter mRNA, especially since the sequence will not actually be transcribed?
I did reporter assay with a revesed 3UTR before (not eactly SV40PolyA); get about 20% singal compare with right direction one.
My best guess is: protein will still expressed with reversed PolyA, (1) since there are mutiple AT rich region in SV40 polyA singal; the direction not affect the transcption machinary meet AT rich site, it is likely there will be a stop and have a tail. There is still arguments between how ployA tail processed in vivo; (2)but within a plasmid and high expression, the stability of the mRNA might het affected, but protein will still translate as the Promoter and stop codon functioning anyway.
Thanks Anyu, after a day of trawling through the literature I realised that they represent the early and late polyadenylation SV40 PolyA signals; early and late genes are transcribed off opposite strands.
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