To study the effect of altered promoter sequences on RNA expression, we performed RT-qPCR for all experiments. The relative Pol II pausing stability (green) and relative RNA expression levels (yellow) are shown. Measurements for the wild-type promoter are normalized to 1, and the relative values for the mutated promoters are shown next to it. Errors bars refer to standard error among two to three biological replicates. Note that the error is large due the variability in transfection efficiency between independent samples. Despite the variability, the change in Pol II pausing stability was in most cases inversely correlated with that of RNA transcription. Thus, a loss in Pol II pausing was usually associated with an increase in transcription. This is consistent with Pol II pausing having an inhibitory effect on transcription. We cannot exclude the possibility that the causality is reverse though, that is that an increase in promoter strength leads to a reduction in Pol II pausing. It is unclear, however, whether promoter strength can be uncoupled from Pol II pausing, and we cannot measure them separately. In our model, promoter strength and Pol II pausing are both determined by TFIID, which binds to both the upstream and downstream core promoter elements we studied.