Reversal of inhibition by the T7 concatemer junction sequence on expression from a downstream T7 promoter.

Abstract

We have previously reported that placement of the phage T7 concatemer junction (CJ) just upstream of another gene on a plasmid in a T7 system proved to be inhibitory to expression of the downstream gene. We had hypothesized that the inhibition was a result of a readthrough transcript of the CJ element interacting with the translation start region of the downstream gene; also that in the absence of a T7 termination signal, transcription continued around the plasmid multiple times ("rolling circle" transcription), always juxtaposing the inhibitory CJ sequence proximal to the downstream gene mRNA. Two strong predictions were made from this model: 1) that introduction of a spacer sequence between the CJ element and the downstream gene should alleviate the inhibition, and 2) that reintroduction of a T7 transcription terminator should prevent rolling circle transcription, thereby reversing the inhibition by allowing some transcripts to be generated originating from the downstream promoter that did not contain the inhibitory CJ element upstream. We report here that both of these predictions have been fulfilled. However, the reversal of inhibition was only partial in the construct where the T7 terminator was reintroduced, indicating that there remains a residual inhibitory effect of the CJ element on expression of the downstream gene. A possible explanation is that the CJ element, acting as a pause site for transcription, blocks access to the downstream T7 promoter, thereby reducing transcription from that promoter. If this explanation is correct, steric hindrance of transcription starts resulting from an upstream RNA polymerase pause site may represent a previously unrecognized mechanism of transcriptional control.