Sequence-Dependent Promoter Escape Efficiency Is Strongly Influenced by Bias for the Pretranslocated State during Initial Transcription.

Abortive transcription initiation can be rate-limiting for promoter escape and therefore represents a barrier to productive gene expression. The mechanism for abortive initiation is unknown, but the amount of abortive transcript is known to vary with the composition of the initial transcribed sequence (ITS). Here, we used a thermodynamic model of translocation combined with experimental validation to investigate the relationship between ITS and promoter escape on a set of phage T5 N25 promoters. We found a strong, negative correlation between RNAP’s propensity to occupy the pretranslocated state during initial transcription and the efficiency of promoter escape (r = −0.67; p < 10–6). This correlation was almost entirely caused by free energy changes due to variation in the RNA 3′ dinucleotide sequence at each step, implying that this sequence element controls the disposition of initial transcribing complexes. We tested our model experimentally by constructing a set of novel N25-ITS promoter variants; quan...