Inhibition of transgene expression in plant protoplasts by the presence in cis of an opposing 3′-promoter

Abstract

The possibility that gene expression could be negatively modulated by RNA polymerases transcribing a gene from its 3′ end towards its 5′ end concurrent with normal 5′ to 3′ transcription has been investigated. The effect of 3′ promoter presence and orientation on 5′ to 3′ gene transcription was determined by comparing transient chloramphenicol acetyltransferase (CAT) activity in Nicotiana tabacum suspension culture protoplasts electroporated with various cat gene containing plasmids. Initial constructs contained the CaMV 35S promoter 5′ to the Tn9 cat gene- nos terminator and in some cases the CaMV 35S promoter 3′ to the nos terminator. Using these constructs in linearized form a statistically significant reduction, about two-fold, in CAT activity as compared to normal single 5′ promoter configuration driven expression, was seen only in the presence of a 3′ promoter inverted with respect to the 5′ promoter. The presence of a 3′ promoter in the same orientation as the 5′ promoter resulted in no significant reduction in CAT activity. Possible mechanisms include antisense RNA production, RNA polymerase ‘colllision’, transcriptionally induced topological constraints and/or destabilization of positive transcriptional factors. The effects of decreasing the 5′ promoter strength and providing a strong polyA-signal/terminator for the antisense strand are also reported.