Abstract
Despite their apparent simplicity, bacteria can do amazing things. They can move, reproduce themselves, send and respond to signals, and perform all the energetic transactions necessary for these tasks. Most of the functions that prokaryotic cells need to survive and proliferate require the coordinated activity of multiple genes. At the most basic level, this coordination is assisted by the organization of functionally related genes into operons. However, often additional control over gene expression exists, in which the activity of multiple operons is coordinated. Functionally-related operons tend to be spatially clustered in the bacterial genome. Moreover, there is evidence that proximally located operons show higher levels of co-expression than far away ones. However, the effect of genomic distance in the global regulation of transcription is still not completely understood. In this thesis, I propose a novel mechanism that could serve to transcriptionally couple neighboring genes. It is based on previous in vitro observations of the behavior of the E. coli RNA polymerase (RNAP) after it has completed transcription of a gene. I present a combination of analytical calculations, stochastic simulations, and single-molecule microscopy experiments used to test the proposed mechanism and characterize the distance and time over which it could couple expression of proximal genes.
Single-molecule microscopy experiments on bacterial transcription are generally performed using immobilized linear DNA templates. However, RNAP binds DNA ends with high affinity, which can result in non-physiological behavior. To avoid such artifacts in our single-molecule experiments, I designed and synthesized fluorescently labeled circular DNA templates for single-molecule fluorescence microscopy. In this thesis, I present the protocols developed for their synthesis. Additionally, I show that the proposed method produces transcriptionally active circular DNA templates that can be used to study the post-termination behavior of RNAP.