Single-molecule analysis of transcription and transcription regulation

Transcription and transcription regulation are complex but important processes. Although the use of conventional biochemical methods has greatly improved our understanding of these processes, these macroscopic techniques have some limitations. First, these techniques often fail to detect unique properties of individual molecules that may be important in understanding their functions. Second, it can be challenging to obtain a complete mechanistic and kinetic understanding of complex processes using ensemble measurements. Application of single-molecule techniques in studying transcription and transcription regulation processes circumvents the limitations of macroscopic methods. In this thesis, I present results from single-molecule studies on two model systems, lactose repressor and RNA polymerase of Escherichia coli. By combining novel and existing microscopy based single-molecule techniques, I have identified a new class of lac repressor mutation that has little effect on mono-operator binding or inducer binding affinity but significantly increases the kinetic stability of repressor-DNA looped complexes. The study also confirms that dissociation of repressor tetramer to dimers plays little role in operator-repressor-operator ternary complex breakdown. Furthermore, these single-molecule studies also demonstrated that lac repressor can undergo large-scale conformational change resulting in formation of structurally distinct but interconvertible looped species. In the transcription part of this thesis, I synthesized a homopolymeric and a copolymeric (which contains alternating nucleotide sequence) DNA templates. Full-length transcripts were produced by RNA polymerase with the copolymeric template. In contrast, transcripts of the homopolymeric template were heterogeneous in length. This indicates that RNA polymerase cannot transcribe long stretch of homopolymeric sequence. In order to learn more about the translocation behavior of RNA polymerase during elongation, I have developed a fluorescent microscopy based single-molecule method that can detect translocation of RNA polymerase at sub-helical turn resolution in real time. Preliminary studies suggest that RNA polymerase can slide back and forth along the DNA template during elongation.