Seminar Series
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Date:
Time: Location: Speaker: Title: |
17 May 17
2-3pm LT7, Hicks Building Dr Achillefs Kapanidis, University of Oxford Mechanisms of bacterial transcription: from single molecules to single cells |
Abstract: The talk will focus on the mechanisms of transcription by the bacterial RNA polymerase (RNAP) at the single-molecule level, both in vitro and in vivo. Using in vitro methods, we recently captured real-time views of initial transcription by RNAP; the mechanism of this process has remained unclear due to the presence of transient intermediates and heterogeneity. To study the kinetics of initial transcription directly, we used single-molecule FRET to observe DNA scrunching and unscrunching on immobilised initial transcribing complexes for several minutes. Our work uncovered extensive RNAP pausing and backtracking during initiation, with region 3.2 of σ70 being an important pausing determinant; such pausing is likely to have important regulatory roles. Current work focuses on the sequence-dependence and mechanism of initiation pausing.
I will also discuss studies of the spatial organisation of transcription at the single-cell level, a feature crucial for gene expression and regulation. Specifically, we use in vivo photo-activated single-molecule tracking to distinguish between diffusing RNA polymerases and RNA polymerases specifically bound to DNA, either on promoters or transcribed genes. We find that transcription can cause spatial reorganisation of the nucleoid, with movement of gene loci out of the bulk of DNA as levels of transcription increase. We also studied the mode of interaction of RNAP with the DNA during promoter search, showing that RNAP interacts substantially with non-specific DNA. Current work focuses on novel ways to study non-specific interactions of DNA-binding proteins with chromosomal DNA, and intracellular diffusion of proteins in the absence of the chromosome. Our work provides a global view of the organisation of transcription and its interplay with chromosome organisation in living bacteria.
I will also discuss studies of the spatial organisation of transcription at the single-cell level, a feature crucial for gene expression and regulation. Specifically, we use in vivo photo-activated single-molecule tracking to distinguish between diffusing RNA polymerases and RNA polymerases specifically bound to DNA, either on promoters or transcribed genes. We find that transcription can cause spatial reorganisation of the nucleoid, with movement of gene loci out of the bulk of DNA as levels of transcription increase. We also studied the mode of interaction of RNAP with the DNA during promoter search, showing that RNAP interacts substantially with non-specific DNA. Current work focuses on novel ways to study non-specific interactions of DNA-binding proteins with chromosomal DNA, and intracellular diffusion of proteins in the absence of the chromosome. Our work provides a global view of the organisation of transcription and its interplay with chromosome organisation in living bacteria.
