The Hidden Genome: How the nonspecific DNA background impacts transcription regulation

Abstract

Transcription is the process by which RNA is synthesized from a DNA template and forms the first step in gene expression. Its regulation depends on interactions between DNA, RNA polymerase, and transcription factors, which determine whether and how strongly a gene is expressed. While many models focus on specific binding sites such as promoters and operators, DNA-binding proteins also interact weakly with the large background of nonspecific DNA present in cells. This nonspecific DNA can affect how much RNA polymerase and transcription factors remain freely available, and therefore influence transcriptional output. This thesis investigates the role of nonspecific DNA in transcription and transcription regulation using a controlled in vitro system. The experimental approach is based on a minimal transcription system in which RNA production is monitored directly with fluorescent RNA aptamers. When the transcribed RNA folds and binds its dye, it produces a fluorescent signal proportional to RNA synthesis. This allows transcription to be measured directly, without relying on translation or protein production. Using the Broccoli RNA aptamer and T7 RNA polymerase, transcription rates are shown to be well described by a thermodynamic promoter-occupancy model: the rate of transcription is proportional to the probability that RNA polymerase occupies the promoter. This framework also describes simple repression by the lac repressor LacI, where different promoter states are weighted according to their statistical probabilities. The resulting fold change in transcription collapses onto a master curve, showing that the same physical principles used to describe transcription regulation in vivo can also explain this simplified in vitro system. The thesis further shows that nonspecific DNA can strongly reduce transcription. Adding increasing concentrations of salmon sperm DNA decreases transcription rates because RNA polymerase partitions onto nonspecific binding sites, leaving less polymerase available to bind the promoter. Nonspecific DNA can therefore act as an effective repressor, even without specific regulatory sequences. When LacI and nonspecific DNA are combined, nonspecific DNA mainly reduces transcription by sequestering RNA polymerase, while only weakly changing the balance between active and repressed promoter states within the tested regime. A theoretical model based on effective protein fugacities explains the data and predicts that nonspecific DNA can either weaken or strengthen repression depending on the relative nonspecific binding affinities of RNA polymerase and repressor. A second finding is that nonspecific DNA strongly delays the appearance of fluorescence. This delay cannot be explained only by reduced transcription rates. Instead, the results support a kinetic model in which newly produced RNA is transiently sequestered by nonspecific DNA in non-fluorescent states, slowing effective aptamer folding. This effect is observed for both Broccoli and the faster folding Okra aptamer, indicating a more general mechanism. Overall, this thesis demonstrates that nonspecific DNA has a measurable and theoretically describable influence on in vitro transcription. It shows that the nonspecific DNA background can shape gene expression by changing the availability of regulatory proteins and by affecting RNA aptamer-based readouts of transcription.