In this work, we study the effect of macromolecular crowding on transcription initiation and report a surprising result: while the first step of promoter opening slows down, the subsequent step of RNA synthesis and promoter escape becomes faster in the presence of crowders. This suggests that the crowded cellular environment significantly impacts gene function, with the effect varying between promoters.

Transcription initiation, the first step in gene expression, has been studied extensively in dilute buffer, a condition which fails to consider the crowded environment in live cells. Recent reports indicate the kinetics of promoter escape is altered in crowded conditions for a consensus bacterial promoter. Here, we use a real-time fluorescence enhancement assay to study the kinetics of unwound bubble formation and promoter escape for three separate promoters. We find that the effect of crowding on transcription initiation is complex, with lower rates of unwound bubble formation, higher rates of promoter escape, and large variations depending on promoter identity. Based on our results, we suggest that altered conditions of crowding inside a live cell can trigger global changes.